Synthetic biomaterial compound of calcium phosphate phases particularly adapted for supporting bone cell activity

ABSTRACT

The present invention is directed to a synthetic biomaterial compound based on stabilized calcium phosphates and more particularly to the molecular, structural and physical characterization of this compound. The compound comprises calcium, oxygen and phosphorous, wherein at least one of the elements is substituted with an element having an ionic radius of approximately 0.1 to 1.1 Å. The knowledge of the specific molecular and chemical properties of the compound allows for the development of several uses of the compound in various bone-related clinical conditions.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of co-pending U.S.divisional application U.S. Ser. No. 09/971,148, filed Oct. 4, 2001,which is a divisional of U.S. Ser. No. 09/044,749, filed Mar. 19, 1998,issued as U.S. Pat. No. 6,323,146 which is a continuation-in-part ofco-pending U.S. patent application Ser. No. 09/029,872 filed Mar. 2,1998, which is a U.S. national phase application of PCT/CA96/00585,filed Aug. 30, 1996, which is a continuation-in-part of U.S. Ser. No.08/576,238, filed Dec. 21, 1995, now abandoned, which claims the benefitof U.S. Provisional Application Serial No. 60/003,157, filed Sep. 1,1995, which are each hereby incorporated herein in their entirety byreference.

FIELD OF THE INVENTION

[0002] This invention is directed to a synthetic biomaterial compoundbased on stabilized calcium phosphates and more particularly to themolecular, structural and physical characterization of this compound.This compound which in the alternative may be referred to as Skelite,™has applications in the treatment of various bone related clinicalconditions such as for the repair and restoration of natural bonecompromised by disease, trauma or genetic influences.

BACKGROUND OF THE INVENTION

[0003] Bone is a complex mineralizing system composed of an inorganic ormineral phase, an organic matrix phase, and water. The inorganic mineralphase is composed mainly of crystalline calcium phosphate salts whilethe organic matrix phase consists mostly of collagen and othernoncollagenous proteins. Calcification of bone depends on the closeassociation between the organic and inorganic phases to produce amineralized tissue.

[0004] The process of bone growth is regulated to meet both structuraland functional requirements. The cells involved in the processes of boneformation, maintenance, and resorption are osteoblasts, osteocytes, andosteoclasts. Osteoblasts synthesize the organic matrix, osteoid, of bonewhich after calcium phosphate crystal growth and collagen assemblybecomes mineralized. Osteocytes regulate the flux of calcium andphosphate between the bone mineral and the extracellular fluid.Osteoclasts function to resorb bone and are essential in the process ofbone remodeling. Disturbing the natural balance of bone formation andresorption leads to various bone disorders. Increased osteoclastactivity has been demonstrated to lead to bone disease characterized bya decrease in bone density such as that seen in osteoporosis, osteitisfibrosa and in Paget's disease. All of these diseases are a result ofincreased bone resorption.

[0005] In order to understand the mechanisms involved which regulatebone cell function, it is important to be able to assess the normalfunction of bone cells and also the degree of perturbation of thisactivity in various bone diseases. This will lead to the identificationof drugs targeted to restore abnormal bone cell activity back to withinnormal levels. Together with the identification of the etiology ofabnormal and normal bone cell activity and the assessment of thisactivity, is the desire and need to develop compositions and methods forthe treatment of abnormal bone cell activity, as a result of disease,surgical removal or physiological trauma all of which lead to bonetissue loss. Therapeutics which provide for the replacement and repairof bone tissue, such as with the use of bone implants, are highlydesired.

[0006] Several groups have attempted to provide compositions suitablefor the therapeutic replacement of bone tissue. U.S. Pat. No. 4,871,578discloses a process for the formation of a non-porous smooth coating ofhydroxyapatite suitable for implant use. U.S. Pat. No. 4,983,182discloses a ceramic implant which comprises a sintered body of zirconiaand a coating of α-TCP and zirconia, or hydroxyapatite and zirconia.U.S. Pat. No. 4,988,362 discloses a composition for the fusion of abioceramic to another bioceramic. U.S. Pat. No. 4,990,163 discloses acoating used for the production of bioceramics which consist of α-TCPand β-TCP. Although these different compositions may be used asbiocompatible coatings for implants and the like, none of thesecompositions have been demonstrated to participate in the natural boneremodeling process. Furthermore, none of the prior compositionsdeveloped, can be manipulated to reliably produce a range of films,thicker coatings and bulk ceramic pieces which share a commoncomposition and morphology which leads to similar bioactive performancein vivo and in vitro.

[0007] It has therefore long been the goal of biomaterials research inthe field of orthopedics to develop synthetic structures exhibitingcomprehensive bioactivity. Bioactive synthetic substrates and scaffoldscapable of incorporation into the natural process of bone remodeling areof interest in applications which include not only in vitro bone cellassays (Davies, J., G. Shapiro and B. Lowenberg. Cells and Materials3(3) 1993; pp. 245-56), but also in vivo resorbable bone cements(Gerhart, T., R. Miller, J. Kleshinski and W. Hayes. J Biomed Mater Res22 1988; pp. 1071-82 and Kurashina, K., H. Kurita, M. Hirano, J.deBlieck, C. Klein and K. deGroot. Journal of Materials Science:Materials in Medicine 6 1995; pp. 340-7), implantable coatings whichenhance the bonding of natural bone to the implant (Tofe, A., G.Brewster and M. Bowerman. Characterization and Performance of CalciumPhosphate Coatings for Implants edited by E. Horowitz and J. Parr.Philadelphia: ASTM, pp. 9-15 (1994), various forms of implantableprostheses and bone repair agents (Tolman, D. and W. Laney. Mayo ClinProc 68 1993; pp. 323-31 and Levitt, S., P. Crayton, E. Monroe and R.Condrate. J Biomed Mater Res 3 1969; pp. 683-5), and ex vivo tissueengineering (Kadiyala,S., N. Jaiswal, S. Bruder. Tissue Engin 3(2) 1997;pp. 173-84). The prime objective for such materials in vivo is tocombine the stimulation of osteogenic activity in associated bonetissues for optimum healing, with the capability to be progressivelyresorbed by osteoclasts during normal continuous remodeling (Conklin,J., C. Cotell, T. Barnett and D. Hansen. Mat Res Soc Symp Proc 414 1996;pp. 65-70). In vitro, related functions are to provide standardizedlaboratory test substrates on which osteoclast resorptive function orosteoblast production of mineralized bone matrix can be assessed andquantified (Davies, J., G. Shapiro and B. Lowenberg. Cells and Materials3(3) 1993; pp. 245-56). Such substrates must be stable and insoluble inthe biological environment until acted upon by osteoclasts, the specificbone mineral resorbing cells.

[0008] While calcium hydroxyapatite (Ca₅(OH)(PO₄)₃ or HA) is the primaryinorganic component of natural bone (Yamashita, K., T. Arashi, K.Kitagaki, S. Yamada and T. Umegaki. J Am Ceram Soc 77 1994; pp. 2401-7),trace elements are also present (Biominerals edited by F. Driessens andR. Verbeeck. Boston: CRC Press (1990). Calcium hydroxyapatite is but oneof a number of calcium-phosphorous (Ca—P) compounds which arebiocompatible. Others include octacalcium phosphate (Brown, W., M.Mathew and M. Tung. Prog Crys Growth Charact 4 1981; pp. 59-87) and bothphases of tricalcium phosphate (Ca₃(PO₄)₂ or α-TCP/β-TCP) (Elliott J.Structure and Chemistry of the Apatites and Other CalciumOrthophosphates New York: Elsevier (1994). Compounds, particularly HA,may show differing degrees of stoichiometry with the Ca/P ratio rangingfrom 1.55 to 2.2 (Meyer, J. and B. Fowler. Inorg Chem 21 1997; pp.3029-35). Such materials can be artificially created by conventionalhigh temperature ceramic processing (Santos, R. and R. Clayton. AmericanMineralogist 80 1995; pp. 336-44) or by low temperature aqueouschemistry (Brown, P., N. Hocker and S. Hoyle. J Am Ceram Soc 74(8) 1991;pp. 1848-54 and Brown, P. and M. Fulmer. J Am Ceram Soc 74(5) 1991; pp.934-40). Most of such artificial materials show good biocompatibility inthat bone cells tolerate their presence with few deleterious effects,and indeed enhanced bone deposition may occur (Ito, K., Y. Ooi. CRCHandbook of Bioactive Ceramics edited by T. Yamamuro, L. Hench and J.Wilson. Boca Raton, Fla.: CRC Press, pp. 39-51 (1990) and Ohgushi, H.,M. Okumura, S. Tamai, E. Shors and A. Caplan. J Biomed Mater Res 241990; pp. 1563-70). Currently, the most recognized medical applicationof calcium phosphates is the coating of implantable prosthetic devicesand components by thermal or plasma spray to render the surfaceosteoconductive. It has been noted that Ca-P ceramics which are stablein biological environments are often a mixture of individual compounds(LeGeros, R., G. Daculsi. CRC Handbook of Bioactive Ceramics edited byT. Yamamuro, L. Hench and J. Wilson. Boca Raton: CRC Press, (1990)).However, despite the osteogenic potential of these artificial materials,none actively participate in the full process of natural boneremodeling.

[0009] In an effort to understand the cellular mechanisms involved inthe remodeling process, several research groups have attempted todevelop methods to directly observe the activity of isolated osteoclastsin vitro. Osteoclasts, isolated from bone marrow cell populations, havebeen cultured on thin slices of natural materials such as sperm whaledentine (Boyde et al Brit. Dent. J. 156, 216, 1984) or bone (Chambers etal J. Cell Sci. 66, 383, 1984). The latter group have been able to showthat this resorptive activity is not possessed by other cells of themononuclear phagocyte series (Chambers & Horton, Calcif Tissue Int. 36,556, 1984). More recent attempts to use other cell culture techniques tostudy osteoclast lineage have still had to rely on the use of corticalbone slices (Amano et al. and Kerby et al J. Bone & Min. Res. 7(3)) forwhich the quantitation of resorptive activity relies upon either twodimensional analysis of resorption pit areas of variable depth or stereomapping of the resorption volume. Such techniques provide at best anaccuracy of approximately 50% when assessing resorption of relativelythick substrata. In addition these analysis techniques are also verytime consuming and require highly specialized equipment and training.Furthermore, the preparation and subsequent examination of bone ordentine slices is neither an easy nor practical method for theassessment of osteoclast activity.

[0010] The use of artificial calcium phosphate preparations as substratafor osteoclast cultures has also met with little success. Jones et al(Anat. Embryol 170, 247, 1984) reported that osteoclasts resorbsynthetic apatites in vitro but failed to provide experimental evidenceto support this observation. Shimizu et al (Bone and Mineral 6, 261,1989) have reported that isolated osteoclasts resorb only devitalizedbone surfaces and not synthetic calcium hydroxyapatite. These resultswould indicate that functional osteoclasts are difficult to culture invitro.

[0011] In the applicant's published international PCT applicationWO94/26872, cell-mediated resorption was shown to occur on a calciumphosphate-based thin film formed by the high temperature processing of acalcium phosphate colloidal suspension on quartz substrates. When usedin vitro, these ceramic films exhibited multiple discrete resorptionevents (lacunae) across their surface as a result of osteoclastactivity, with no evidence of dissolution arising from the culturemedium. The regular margins of these lacunae correspond closely to thesize and shape of the ruffled borders normally produced by osteoclastsas the means by which they maintain the localized low pH required tonaturally resorb bone mineral in vivo. Enhanced deposition ofmineralized bone matrix also occurs on these ceramics in the presence ofosteoblasts.

[0012] It is now demonstrated by the Applicant's that these thin filmceramics exhibit two general characteristics: (1) the presence of amixture of Ca—P containing phases comprising approximately 33% HA andapproximately 67% of a silicon stabilized calcium phosphate and (2) aunique morphology. Importantly, it was noted that the thermal processingof the Ca—P colloid at 1000° C. resulted in an HA powder, while the samecolloidal suspension processed on quartz had a mixed HA and siliconstabilized calcium phosphate phase composition. Energy dispersive X-rayanalysis of the film demonstrated the presence of Si in the coatingwhile cross-sectional transmission electron microscopy indicated amicroporous physical structure.

[0013] Applicants have discovered that the presence of stabilizingentities can stabilize the composition and prevent its degradation inphysiological fluids. Hence, disappearance of calcium phosphate entitiesfrom a film, coating or bulk ceramic piece of this composition, issubstantially due to the activity of the osteoclasts and not due to adissolution process. The stabilized artificial bioactive composition isthe first such composition which supports both osteoclast and osteoblastactivity and which allows for the reliable assessment of thephysiological activities of both cell types as well as for thedevelopment of both diagnostic and therapeutic strategies.

[0014] In view of the clinical importance of developing a synthetic bonegraft that is both osteogenic and can participate in the body's naturalcell-based remodeling process, it was important to focus on the role ofintroduced additives such as silicon in the formation of a calciumphosphate-based biomaterial compound capable of being assimilated andremodeled into natural bone with the aid of the activity of osteoclastsand osteoblasts. Since the compound could only be characterized by thepreparation method, it was crucial to be able to both physically andchemically characterize the compound. In particular, it was important tocharacterize the physical structure of the compound and moreimportantly, the specific molecular and chemical structure of thestabilized compound in order to be able to understand why the newcompound worked so well in biological conditions affecting the skeleton.The physical, molecular and chemical characterization of the compoundcould also provide for the development of further uses of the compoundin the treatment of several different types of bone-related clinicalconditions. In addition, this would also allow further chemicalalteration of the compound in order that it could be designed for use inspecific in vivo, in vitro and ex vivo applications.

[0015] The Applicant's work now pointed to the transformation of HA intoa stabilized calcium phosphate phase. Surprisingly, during the difficultcourse of explicit characterization of the compound from a molecularstandpoint, it was found-that the resultant stabilized compound was anentirely new compound herein described and termed Skelite™.

SUMMARY OF THE INVENTION

[0016] The present invention provides a stabilized compositioncomprising a synthetic biomaterial compound which allows for a widevariety of diagnostic and therapeutic applications. The biomaterialcompound, in accordance with an aspect of the invention, can be used toprovide a range of fine or coarse powders, pellets, three-dimensionalshaped pieces, thin films and coatings which share a common globularmorphology and an interconnected microporosity. In addition, thebiomaterial compound can be formed as a macroporous structure in orderto provide an artificial three dimensional geometry similar to thatfound in bone in vivo. The biomaterial compound, made in any form,encourages the activity of bone cells cultured thereon and also allowsfor the development of ex vivo engineered artificial bone tissues foruse as bone grafts.

[0017] The created stabilized calcium phosphate compound has only nowbeen specifically characterized with respect to its physical andchemical structure leading to the realization that the stabilizedcompound was an entirely new compound never before described. Thebiomaterial compound is made by the high temperature processing of afine precipitate, formed from a colloidal suspension and stabilizedusing an additive with an appropriate sized ionic radius that enablessubstitution into the Ca—P lattice. The compound typically coexists withcalcium hydroxyapatite and is itself a novel stabilized calciumphosphate compound having a microporous morphology based oninter-connected particles of about 0.2-1.0 μm in diameter. The compoundis essentially insoluble in biological media but is resorbable whenacted upon by osteoclasts. It also promotes organic bone matrixdeposition by osteoblasts and can be assimilated into natural boneduring the natural course of bone remodeling through the activity ofosteoclasts and osteoblasts. The compound has been extensively analyzedusing X-ray diffraction, infrared spectroscopy, nuclear magneticresonance spectroscopy, and light scattering particle analysis. Resultsnow indicate that the characteristic features of the compound ariseduring sintering through substitution reactions where a stabilizingelement such as silicon enters the calcium phosphate lattice underconditions of high chemical reactivity. The crystallographic featuresare linked through the glaserite form of the apatite structure.

[0018] According to an aspect of the present invention a biomaterialcompound is provided comprising calcium, oxygen and phosphorous, whereinat least one of the elements is substituted with an element having anionic radius of approximately 0.1 to 1.1 Å.

[0019] According to another aspect of the present invention is abiomaterial compound having the formula(Ca_(1-w)A_(w))_(i)[(P_(1-x-y-z)B_(x)C_(y)D_(z))O_(j)]₂; wherein A isselected from those elements having an ionic radius of approximately 0.4to 1.1 Å; B, C and D are selected from those elements having an ionicradius of approximately 0.1 to 0.4 Å; w is greater than or equal to zerobut less than 1; x is greater than or equal to zero but less than 1; yis greater than or equal to zero but less than 1; z is greater than orequal to zero but less than 1; x+y+z is greater than zero but less than1; i is greater than or equal to 2 but less than or equal to 4; and jequals 4-δ, where δ is greater than or equal to zero but less than orequal to 1.

[0020] Specific compounds of the present invention include but are notlimited to Ca₃(P_(0.750)Si_(0.25)O_(3.875))₂ andCa₃(P_(0.9375)Si_(0.0625)O_(3.96875))₂.

[0021] The knowledge of the specific molecular and chemical propertiesof the compound of the present invention allows for the development ofseveral uses of the compound in various bone-related clinicalconditions. Such applications may include orthopedic, maxillo-facial anddental applications where the compound can be fabricated to exist as afine or coarse powder, pellets, three-dimensional shaped pieces,macroporous structures, thin films and coatings.

[0022] According to yet another aspect of the present invention is amethod for substituting natural bone at sites of skeletal surgery inhuman and animal hosts with a biomaterial compound comprising calcium,oxygen and phosphorous wherein at least one of the elements issubstituted with an element having an ionic radius of approximately 0.1to 1.1 Å. The method comprises the steps of implanting the biomaterialcompound at the site of skeletal surgery wherein such implantationpromotes the formation of new bone tissue at the interfaces between thebiomaterial compound and the host, the progressive removal of thebiomaterial compound primarily through osteoclast activity, and thereplacement of that portion of the biomaterial compound removed byfurther formation of new bone tissue by osteoblast activity, suchprogressive removal and replacement being inherent in the natural boneremodeling process.

[0023] In accordance with another aspect of the present invention is amethod for repairing large segmental skeletal gaps and non-unionfractures arising from trauma or surgery in human and animal hosts usinga biomaterial compound comprising calcium, oxygen and phosphorouswherein at least one of the elements is substituted with an elementhaving an ionic radius of approximately 0.1 to 1.1 Å. The methodcomprises the steps of implanting the biomaterial compound at the siteof the segmental skeletal gap or non-union fracture wherein suchimplantation promotes the formation of new bone tissue at the interfacesbetween the biomaterial compound and the host, the progressive removalof the biomaterial compound primarily through osteoclast activity, andthe replacement of that portion of the biomaterial compound removed byfurther formation of new bone tissue by osteoblast activity, suchprogressive removal and replacement being inherent in the natural boneremodeling process.

[0024] According to yet another aspect of the present invention is amethod for aiding the attachment of implantable prostheses to skeletalsites and for maintaining the long term stability of the prostheses inhuman and animal hosts using a biomaterial compound comprising calcium,oxygen and phosphorous wherein at least one of the elements issubstituted with an element having an ionic radius of approximately 0.1to 1.1 Å. The method comprises the steps of coating selected regions ofan implantable prosthesis with the biomaterial compound, implanting thecoated prosthesis into a skeletal site wherein such implantationpromotes the formation of new bone tissue at the interfaces between thebiomaterial compound and the host, the generation of a secureinterfacial bond between the host bone and the coating, the subsequentprogressive removal of the coating primarily through osteoclast activitysuch that the coating is diminished, and the replacement of that portionof the biomaterial compound removed by further formation of new bonetissue to generate a secure interfacial bond directly between the hostbone and the prosthesis.

[0025] According to yet another aspect of the present invention is amethod for providing tissue-engineering scaffolds for bone replacementin human or animal hosts using a biomaterial compound comprisingcalcium, oxygen and phosphorous wherein at least one of the elements issubstituted with an element having an ionic radius of approximately 0.1to 1.1 Å. The method comprises the steps of forming the biomaterialcompound as a macroporous structure comprising an open cell constructionwith interconnected voids, combining mature and/or precursor bone cellswith the macroporous structure, and allowing the cells to infiltrate thestructure in order to develop new mineralized matrix throughout thestructure.

[0026] The knowledge of the structure of the novel compound of thepresent invention also allows for the use of the compound as a carrierfor various pharmaceutical agents including but not restricted to bonegrowth factors and other agents affecting bone growth and remodeling.

[0027] According to another aspect of the present invention is a methodfor delivering pharmaceutical agents to the site of skeletal surgery inhuman or animal hosts using a biomaterial compound comprising calcium,oxygen and phosphorous wherein at least one of said elements issubstituted with an element having an ionic radius of approximately 0.1to 1.1 Å. The method comprises combining a pharmaceutical agent with thebiomaterial compound and applying the pharmaceutical agent combined withthe biomaterial compound to a site of skeletal surgery, wherein suchapplication results in controlled local release of the pharmaceuticalagent.

[0028] The biomaterial compound may be combined with additives such asthose which increase the mechanical strength and toughness of thecompound in order to provide additional functions for specificapplications. The biomaterial compound may also be combined with variouscalcium materials such as calcium hydroxyapatite, α-TCP, β-TCP,octocalcium phosphate, tetracalcium phosphate, dicalcium phosphate andcalcium oxide either as a physical mixture or as a solid solution.

[0029] The biomaterial compound has a distinguishable microporous andnanoporous structure along with a crystallography that is similar yetdifferent from that of α-TCP. The new compound exhibits monoclinicpseudo-rhombic symmetry and is in the monoclinic space group P2₁/a.Furthermore, the new compound has a portion of the phosphoroussubstituted by an element having a suitable ionic radius.

[0030] The knowledge of the chemical formula of the biomaterial compoundand the mechanism behind its bioactivity and stability in biologicalenvironments allows for the use of this compound in vivo for thetreatment of various bone related clinical conditions. In particular,the compound may be used to help repair and restore natural bone thathas been compromised by disease, trauma, or genetic influences.

[0031] In accordance with yet a further aspect of the invention is abioactive synthetic sintered composition for providing a morphologycapable of consistently supporting bone cell activity thereon, thecomposition comprising stabilized calcium phosphate compound developedby the conversion of a hydroxyapatite substance in the presence ofstabilizing entities at sintering temperatures wherein the stabilizingentities stabilize and insolubilize the calcium phosphate compound.

[0032] In accordance with a further aspect of the present invention is aprocess for preparing a synthetic sintered composition comprising astabilized calcium phosphate compound having a morphology suitable forsupporting bone cell activity thereon, the process comprising convertinga hydroxyapatite substance in the presence of stabilizing entities atsintering temperatures wherein the stabilizing entities stabilize andinsolubilize the calcium phosphate compound.

[0033] According to yet a further aspect of the present invention is asynthetic sintered microporous polycrystalline structure for supportingbone cell activity, the structure comprising a stabilized calciumphosphate compound having a globular morphology of interconnectedrounded particles with an interconnected microporosity in saidstructure.

[0034] In accordance with yet a further aspect of the present inventionis an implant comprising: a) a scaffold for supporting the implant; andb) a layer of a stabilized calcium phosphate compound developed by theconversion of a hydroxyapatite substance in the presence of stabilizingentities at sintering temperatures wherein the stabilizing entitiesinsolubilize and stabilize the calcium phosphate compound.

[0035] In accordance with another aspect of the present invention is animplant comprising: a) a scaffold for supporting the implant; b) a layerof a stabilized calcium phosphate compound developed by the conversionof a hydroxyapatite substance in the presence of stabilizing entities atsintering temperatures wherein the stabilizing entities insolubilize andstabilize the calcium phosphate compound; c) a boundary layer depositedby osteoblasts cultured on the layer of the stabilized calcium phosphatecompound; and d) a mineralized collagenous matrix secreted by suchcultured osteoblasts.

[0036] According to another aspect of the present invention is a methodfor the culturing of functional bone cells, the method comprisingapplying a suspension of bone cells in physiological media to asynthetic sintered film comprising a stabilized calcium phosphatecompound on a substrate; and incubating the bone cells for a period oftime to allow expression of bone cell biological activity.

[0037] According to a further aspect of the present invention is a kitfor monitoring and quantifying the activity of bone cells, the kitcomprising a substrate having a sintered film of a stabilized calciumphosphate compound and a multiwell bone cell culture device adhered tothe substrate.

[0038] In accordance with another aspect of the present invention is amethod for the production of a calcium phosphate biomaterial powder, themethod comprising:

[0039] (a) mixing a silica colloid with a calcium phosphate colloidalsuspension;

[0040] (b) spray drying (a) into powder;

[0041] (c) sintering said powder.

[0042] In an aspect of the invention, the silica colloid is a finelydivided fumed silica colloid.

[0043] In accordance with yet another aspect of the present invention isa method for the production of a calcium phosphate biomaterial powder,the method comprising:

[0044] (a) mixing a solution of calcium nitrate tetrahydrate withammonium dihydrogen orthophosphate to produce a calcium phosphatecolloidal suspension;

[0045] (b) mixing (a) with a finely divided fumed silica colloid;

[0046] (c) spray drying (b) into a powder; and

[0047] (d) sintering using a heating profile with a ramp rate of about5° C. per minute, drying at about 200° C. for about 180 minutes,calcining at about 550° C. for about 60 minutes and sintering at about1000° C. for about 60 minutes.

[0048] The silica colloid may be added at any time in the process andpreferably, prior to the aging and centrifugation of the calciumphosphate colloidal suspension.

[0049] According to another aspect of the invention is a powderedcalcium phosphate biomaterial made by the method recited herein andcontaining up to about 10 weight percent of silicon.

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The present invention will be further understood from thefollowing description with reference to the Figures, in which:

[0051]FIG. 1 shows a X-Ray Diffraction Spectrum (θ-2θ) of powderprepared from the Ca—P colloid with no introduced additives and sinteredat 1000° C.

[0052]FIG. 2 shows glancing angle XRD spectra of a thin film of the Ca—Pcolloid sintered on quartz at 1000° C.

[0053]FIG. 3 shows GA-XRD spectra illustrating the effect of sinteringtemperature on thin film phase composition;

[0054]FIG. 4 shows GA-XRD spectra illustrating the effect of sinteringtime on thin film phase composition;

[0055]FIG. 5 shows a SEM micrograph illustrating the characteristicsurface morphology of a thin film of the Ca-P colloid sintered on quartzat 1000° C.;

[0056]FIG. 6 is a cross-sectional TEM of a Ca-P thin film on quartz, (a)film sintered at 1000° C. (b) unsintered film; (c) EDX analysis of thesintered TEM sample at (i) the interface of the film and the substrate,(ii) the intermediate region above the interface, and (iii) the top ofthe film.

[0057]FIG. 7 shows the average agglomerate size in the Ca—P colloid as afunction of colloid aging period, as determined using light scatteringparticle analysis;

[0058]FIG. 8 shows a calculated predominance area diagram illustratingthe effect of CaO activity on the relative stabilities of HA and TCP;

[0059]FIG. 9 shows a θ2θ XRD spectrum of powder prepared from the Ca—Pcolloid with silicon as the introduced additive. Approximate phaseratio: 33±5% HA and 67÷5% Si-TCP;

[0060]FIG. 10 shows the effect of silicon content on phase compositionof Si-mHA powders, as determined by x-ray diffraction (θ-2θ);

[0061]FIG. 11 shows SEM micrographs illustrating the characteristicsurface morphology of Si-mHA ceramic pellets. Si-mHA pellets can beresorbed by the specific cellular activity of osteoclasts in a mannersimilar to that which occurs on natural bone. (a) Surface morphologySi-mHA ceramic pellet; (b) Osteoclast lacunae on surface of Si-mHAceramic pellet; and 11(c) Osteoclast lacunae on surface of natural bone;

[0062]FIG. 12 shows θ-2θ XRD spectra of powder prepared from the Ca—Pcolloid with titanium as the introduced additive.

[0063]FIG. 13 shows the effect of Ti addition on mHA phase composition;(a) no carrier (powder), (b) no carrier (ceramic pellet), (c) 2Me(powder), (d) 2Me (ceramic pellet), (e) ACAC (powder) and (f) ACAC(ceramic pellet);

[0064]FIG. 14 shows SEM micrographs comparing the microstructure ofSi-mHA pellets formed from the Ca—P colloid versus materials preparedfrom commercial sources. (a) Si-mHA prepared using TPOS as theintroduced additive; and (b) cHA as a physical mixture with TPOS.

[0065]FIG. 15 shows the XRD spectra for the physical mixture of 25%CaSiO₃ and 75% α-TCP sintered at 1250° C. for 8 hours;

[0066]FIG. 16 shows a high resolution XRD spectrum of Si-mHA powder;

[0067]FIG. 17 shows the NMR spectra comparing Si-mHA with commerciallyavailable reference materials; (a) mixture of commercial CaSiO₃ and SiO₂powders, (b)Si-mHA powder;

[0068]FIG. 18 shows IR spectra for powders sintered at 1000° C.: (a)cHA, (b) mHA and (c) Si-mHA; and

[0069]FIG. 19 shows a summary of the IR spectra illustrating the effectof silicon content on the P—O stretch.

[0070]FIG. 20 is a cross-sectional SEM micrograph illustratingmineralized collagenous matrix deposited on a thin film of thestabilized composition.

[0071]FIG. 21 are photographs of fluorescence analysis (a) is thedeposition of fluorescent mineralized matrix produced by osteoblastscultured on the stabilized composition, (b) is a control in which noosteoblasts are cultured on the stabilized composition and nofluorescent mineralized matrix is visualized.

[0072] FIGS. 22(a) and (b) are SEM micrographs of osteoclast resorptionpits on thin films of the stabilized composition.

[0073]FIG. 23 is a micro CT image of natural bone architecture withinset SEM micrograph of a macroporous structure formed from the Skelite™biomaterial compound.

[0074] In the drawings, preferred embodiments of the invention areillustrated by way of example. It is to be expressly understood that thedescription and drawings are only for the purpose of illustration and asan aid to understanding, and are not intended as a definition of thelimits of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0075] The Applicants have developed a process to provide a stabilizedcalcium phosphate synthetic biomaterial compound which is fullybiocompatible and has a morphology capable of consistently supportingbone cell activity thereon. This is provided in accordance with thatmethod described in the Applicant's co-pending published PCT applicationWO 94/26872, the subject matter of which is incorporated herein byreference. The preferred embodiment for making the compound of thepresent invention is described herein in the accompanying examples.

[0076] The compound of the present invention is herein referred to as abiomaterial compound due to its bioactive nature in both in vitro and invivo systems. Bioactivity refers to the ability of the biomaterialcompound to support osteoclast and osteoblast activity and the abilityto be assimilated with natural bone by the activity of these cells.Although the compound was characterized with respect to the process bywhich it is made, both the physical and molecular structure were unknownand could not be determined. It was however essential to characterizethe compound further with respect to its physical and chemical structureso as to better understand the properties of the compound as well as tounderstand why the compound was so well adapted for osteoclast andosteoblast activity. The knowledge of the chemical structure of thecompound also allows for the modification of the compound fortherapeutic use in the treatment of certain clinical conditions.

[0077] Early data pointed to the fact that that the compound was siliconstabilized. With further difficult and tedious analysis it wassurprisingly found that the compound was in fact a completely newsynthetic stabilized calcium phosphate compound never beforecharacterized and herein referred to as Skelite™. Where silicon is usedas the introduced additive to form Skelite™ the compound is referred toas Si-TCP. One reason for the great difficulty in establishing thechemical formula for the new compound was due to the complex and largestructure of Ca—P compounds such as HA as well as the changing phasetransitions that occurred during the sintering process. The chemicalidentification of this compound was only realized and developed afterlengthy analysis of various Ca—P powders, thin films and pelletsprepared with introduced additives. Standard XRD analysis was performedon samples prepared by a variety of compositional and thermal processingroutes. Results were initially considered to be consistent with theconclusion that the materials were a mixture of α-TCP and HA, and thatthe calcium silicates predicted by the FACT database (Bale, C. W., A. D.Pelton, and W. T. Thompson. FACT Database [computer program]. Contact:W. T. Thompson, Chemical and Materials Engineering, Royal MilitaryCollege, Kingston, Canada, K7K 5L0 (1997) existed as a glassy phase atthe grain boundaries. Since no JCPDS file was available for Skelite™ andthe peak positions were indicative of α-TCP using standard XRDtechniques, the identification of Skelite™ was unforeseen. Furthermore,one would not expect to find that substitution is taking place at suchlow temperatures. A complex and unobvious combination of analysistechniques had to be performed to successfully identify and characterizethe new compound. These studies, described as follows led to thecharacterization of the new compound, an additive stabilized calciumphosphate compound, Skelite™.

[0078] For clarity, several materials referred to herein are defined asfollows. For the commercially available materials cHA refers tocommercial calcium hydroxyapatite (HA), calcium silicate refers toCaSiO₃ and silica refers to SiO₂. For the internally prepared materialsmHA refers to microporous calcium hydroxyapatite (HA), Si-mHA refers toSi-TCP plus mHA. These materials are further defined in Table 1.

[0079] Analysis of Pure (No Introduced Additives) mHA Powders

[0080] Using reaction (1) and analogous reactions, a fine colloidalprecipitate of HA in ammoniated water can be achieved under conditionswhere the pH is greater than 10.

5Ca(NO₃)₂+3NH₄H₂PO₄+7NH₄OH Ca₅(OH)(PO₄)₃+10NH₄NO₃+6H₂O  (1)

[0081]FIG. 1 shows that powders prepared from the colloidal suspensionof equation (1) with no introduced additives and sintered at 1000° C.are HA (JCPDS File #9-432). The particle size of sintered powders, aftermild grinding following sintering, is about 1 μm as determined by SEM.

[0082] Analysis of Thin Films on Quartz Substrates

[0083]FIG. 2 shows that the film on quartz has a crystallographicstructure which was more complex than that for a powder sintered underthe same conditions. The structure consists of two major phases, HA andSi-TCP, where the Si-TCP resembles, but is different from thecrystallography of α-TCP (JCPDS file #9-348). All peaks within the XRDspectra could be attributed to either HA or Si-TCP and no distributionsof peaks characteristic of other phases (such as β-TCP or octacalciumphosphate) were distinguishable from background.

[0084]FIG. 3 shows that as the sintering temperature was increased, thefilm composition changed. When the film was fired for one hour at 800°C. the composition of the film was 94% HA and 6% Si-TCP; at 900° C.there was a mixture of 62% HA and 38% Si-TCP; at 1000° C. thecomposition was 33% HA and 67% Si-TCP. Changes in composition and filmmorphology as a function of sintering duration were assessed by changingthe time the thin film on quartz remained in a furnace maintained at aset temperature. A computer controlled system allowed the ramp rate andhold temperature to be defined. FIG. 4 shows that a dwell time of fiveminutes yielded the same equilibrium phase composition as observed aftera one hour dwell time. Increased dwell time resulted in grain growth, asshown by SEM studies.

[0085] The phase composition could be modified by changing the humidityof the sintering environment while maintaining the firing conditions at1000° C. for one hour. The reaction was suppressed by the presence ofincreased water vapor. Other external factors or the addition ofadditives to the colloid suspension did not significantly modify theresults achieved for thin films on quartz.

[0086] Optical microscopy, SEM and TEM show that the sintered films onquartz have a consistent morphology which is illustrated in FIGS. 5 and6(a). While the films appear to be composed of translucent polycrystalsunder an optical microscope with phase contrast (×20), at the highermagnification achieved using an SEM (×10K) the surface morphology isthat of an interconnected set of rounded particles with a high degree ofporosity as seen in FIG. 5. The average dimension of these particlesdepends on the time and temperature of sintering. Under most conditionsthe mean size lies between 0.2 and 1 μm with the size increasing withthe time and temperature of sintering. Cross-sectional TEM (FIG. 6(a))of an individual particle indicates the presence of nanoporosity withinthe body of the particle. It is important to note that these pores werenot altered with extended exposure to the electron beam, and weretherefore inherent to the sample and not a sample preparation artifact.The under lying granular structure was about 5-10 nm in size. Thisappeared to reflect the individual granule size observed in thecross-sectional TEM micrographs of a dried but unsintered thin film onquartz as seen in FIG. 6(b).

[0087] To examine the evolution of particle agglomeration, aliquots ofthe colloidal suspension were analyzed for particle size after variousaging times. FIG. 7 shows that a marked variation in measured particlesize occurs during the 24 hour period of aging. The initial measurementgives a particle size less than 1 μm, increasing to greater than 10 μmafter 8 hours, but subsequently decreasing again to approximately 1 μmafter 24 hours. This is indicative of agglomeration of the fineprecipitate with the most stable structure having dimensions in therange of 0.2-1.0 μm. Subsequent sintering of such agglomerates accountsboth for the basic morphology of the thin films on quartz and themicroporosity of bulk ceramics.

[0088] In order to understand the origin of the difference between aprecipitate fired as a powder or as a thin film prepared on a quartzsubstrate, films on quartz were fired for 1 hour and were subsequentlyanalyzed for elemental composition as a function of distance from thequartz interface using electron induced energy dispersive X-rayspectroscopy (EDX). As shown in FIG. 6(c)(i)(ii) and (iii) silicon wasdetected at concentrations which decreased with distance from theinterface; however, no XRD peaks for compounds such as calcium silicatecould be identified. These results suggested that Si diffusing from thequartz substrate played a role in modifying the morphology andcrystallography of the thin films.

[0089] Analysis of mHA Powders with Introduced Additives

[0090] Powders prepared from the colloid of equation (1) combined withselective additives, demonstrate a unique calcium phosphate compositionafter sintering at 1000° C. Several possible actions of silicon as anadditive in this temperature range were initially postulated such as themodification of the conversion reactions of HA into its successorcompounds; the modification of the crystallographic structure of HA andits successor products by silicon substitution; and morphologicalchanges associated with surface diffusion of the additive or by additiveinduced changes in surface properties.

[0091] These possibilities were evaluated by the creation of ceramicthin films, powders and bulk materials in which processing conditions orthe presence of additives changed the final products. The initial basisfor defining process changes and additive selection was determinedaccording to equilibrium thermodynamic computations using the databaseand programming in the Facility for the Analysis of ChemicalThermodynamics (FACT) (Bale, C. W., A. D. Pelton, and W. T. Thompson.FACT Database [computer program]. Contact: W. T. Thompson, Chemical andMaterials Engineering, Royal Military College, Kingston, Canada, K7K 5L0(1997). FIG. 8 shows the calculated phase diagram expected for the Ca—Psystem as a function of inverse temperature (K⁻¹) and partial pressureof H₂O in the thermal processing atmosphere. The diagram applies to aclosed chemical system and utilizes a large database of literaturevalues for the Gibbs free energies of formation. The most stablephase(s) are computed for a large matrix of coordinates which lead tothe placement of the phase boundaries. HA decomposes into β-TCP attemperatures below 1100° C. under low partial pressure of H₂O. α-TCP isformed at temperatures above about 1100° C. The predictions areconsistent with high temperature crystallographic data for HA ceramics(Welch, J. and W. Gutt. J Chem Soc 1961; pp. 4442-4 and Schroeder, L.,B. Dickens and W. Brown. J Solid State Chem 22 1977; pp. 253-62). Thedecomposition reaction, corresponding to the lowest diagonal line on thediagram, may be written as equation (2):

2Ca₅(OH)(PO₄)₃ _(→) ^(←)3Ca₃(PO₄)₂+CaO+H₂O  (2)

[0092] Since the conversion of HA into TCP results in the simultaneousformation of CaO and release of H₂O, changes in the activity of both CaOand H₂O should modify the location of the phase boundaries. The upperdiagonal lines show the phase boundary when the activity of CaO is madeprogressively smaller. This effect can be practically accomplished bychemical combination of CaO with other compounds such as SiO₂. In thepresence of silica (SiO₂), the resultant compound could be one or moreof several calcium silicates. The calculations show that thedecomposition boundaries in the temperature range of 800-1100° C. are inapproximate agreement if CaO has the activity expected when the CaO iscombined with SiO₂ as follows in equation (3):

CaO+SiO_(2 →) ^(←)CaSiO₃  (3)

[0093] The most stable phosphorous-containing conversion product is,however, β-TCP. This is consistent with the widespread observation ofthe magnesium doped HA-based mineral whitlockite as the natural form ofβ-Ca₃(PO₄)₂ (Schroeder, L., B. Dickens and W. Brown. J Solid State Chem22 1977; pp. 253-62). On the basis of the information available withinthe FACT database it is not possible to explain the observation of aphase similar to α-TCP as a conversion product below 1000° C. other thanto assume that β-TCP is not nucleated when CaSiO₃ forms and that Si-TCPdevelops as a metastable allotropic form.

[0094] It may be noted that on the basis of chemical thermodynamics, anyreaction which changes the activity of CaO should modify the phasediagram. Oxides such as TiO₂ have only one product with CaO as inequation (4):

CaO+TiO_(2 →) ^(←)CaTiO₃  (4)

[0095] and may therefore be more predictable in their action. Similarcalculations to those for Si showed that for a similar partial pressureof water, the phase boundary for Ti was located at a slightly lowertemperature.

[0096]FIG. 9 shows that the XRD pattern for a powder prepared using anadditive concentration of 1 mol SiO₂ to 1 mol mHA is similar to thatobtained for thin films on quartz. For this sample, the silicon wasadded as tetrapropyl orthosilicate in 2-methoxyethanol. The spectrum wascompared to JCPDS files and concluded to be a mixture of HA and Si-TCP.Subsequent experiments demonstrated that the phase composition wasindependent of whether the additive was introduced with2-methoxymethanol, 2-4 pentanedione or no carrier. FIG. 10 shows thephase composition of powders sintered at 1000° C. for one hour as afunction of silicon content, as determined by XRD. The phases presentswitch from predominantly HA to predominantly a new compound (Si-TCP) ata relative molar Si/mHA ratio of approximately 0.6. The conversion isslightly greater when powders are formed into ceramic pellets. While thespecific level of conversion is dependent on processing conditions, thetypical Si-TCP:HA range is 20:80 to 80:20. Due to the increased signalto noise ratio and a more linear change of the background as a functionof 2θ evident in the θ-2θ XRD spectra of the powders, the accuracy ofdetermination of the phase composition in powders is increased. Additivesaturation is evident at molar ratios exceeding 1:1 indicating processconstraints in the integration of further silicon. FIG. 11(a) shows thatthe crystalline morphology of a pellet formed from Si-mHA was similar tothat observed in the thin films on quartz. The ceramic comprisesrounded, inter-connected particles of average size 0.2-1.0 μm with alarge degree of localized porosity. Varying the compound preparationcondition permits the formation of a range of microporous structurescomprised of particles of size range 0.1 to 2.0 μm. FIG. 11(b) indicatesthat Si-mHA materials show strong evidence of osteoclastic resorptionsimilar to that which occurs on natural bone as shown in FIG. 11(c).

[0097] The XRD pattern for powders prepared using Ti as the additivealso showed that conversion occurs upon addition of the Ti. However, theresults were more complex as the predominant phase of TCP formed wasβ-TCP (FIG. 12) with the degree of conversion dependent on the carrierused with the additive. Furthermore, there was enhancement of the degreeof conversion on powder grinding and processing to form ceramic pellets.The results are summarized in FIG. 13. FIGS. 13(a) and 13(b)respectively show the effects of titanium addition with no carrier forpowders and ceramic pellets. Substantial conversion only occurs forpellets which were formed by grinding, pressing and resintering theoriginal powder. The addition of titanium is similar or even lesseffective when 2Me is used as the carrier (FIGS. 13(c) and 13(d)).Substantial conversion at approximately 0.5 mol TiO₂ per mol mHA occursin powders only when ACAC is used as a carrier and again this conversionoccurs more effectively in the reground pellets, FIGS. 13(e) and 13(f).Particularly in the ceramic pellets, the phase composition shows asubstantial fraction of β-TCP. The microstructure of pellets createdfrom powders where Ti was the additive showed a particle size ofapproximately 0.3 μm.

[0098] The simplest interpretation of the differences between theeffects of Si and Ti additives is based on the observation of theeffects of additive precipitation and the changes observed in degree ofconversion following powder grinding and pellet formation. In the caseof Si-based additions, the degree of precipitation was essentiallyindependent of the carrier and relatively minor changes in the degree ofconversion occurred on formation into ceramic pellets. In contrast, Tiadditions were ineffective when precipitation occurred when the additivewas introduced into the Ca—P colloidal suspension (for no carrier and2Me). Ti additions were effective when precipitation did not occur (forACAC) and conversion became stronger upon grinding of the powder to formpellets and subsequent resintering. This suggests that the conversionfrom HA to TCP requires intimate contact between the additive and HA,possibly through surface functionalization of the precipitated mHAparticles within the colloid suspension by the additive species oradsorption of the additive species on the surface of the mHA particle.When the additive and the mHA precipitate as separate species, theconversion occurs only upon strong physical inter-mixing and thermaltreatment.

[0099] For comparative purposes, reference materials were prepared byequivalent thermal processing of commercially available powders (seeTable 1) in an attempt to produce ceramics with a similar phasecomposition and surface morphology. Commercial powders were processed aspure compounds and in combination with selective additives introducedeither as inorganic powders or as metallorganic species in a carrier.XRD results indicate that conversion of commercial HA (cHA) does takeplace, but that the primary resultant phase is β-TCP. The typical phasedistribution is 73% β-TCP, 20% α-TCP and 7% HA. These results areconsistent with the phase composition predicted by thermodynamics asnoted in equations (2) and (3) and illustrated in FIG. 8. Of equalsignificance is that the surface morphology of the ceramics preparedfrom these powders exhibits a jagged or fractured morphology (FIG. 14b)with little inter-connectedness, and a particle size an order ofmagnitude greater than observed in colloid-based mHA pellets (FIG. 14a).Evidence for a microporous morphology is restricted to the surfaceregion of the particles. Pellets prepared in this fashion show noindication of resorption by osteoclasts.

[0100] The solid state chemistry of the cHA powders with introducedadditives suggest that the conversion behaviour as a function oftemperature, humidity and additive is consistent with equations (2)-(4).In particular, if physical mixing of the additive into the cHA powderstakes place the β-TCP phase predicted by chemical thermodynamics isobserved. In comparison, if intimate mixing of an unprecipitated siliconadditive and a Ca—P colloid occurs the resultant phase is Si-TCP. Thisphase is not consistent with the predictions of equilibriumthermodynamics, but it is closely linked with the presence of Si in theCa—P lattice. In order to use the FACT database to predict the phaseboundaries for transitions to this Skelite™ compound, new values for theGibbs free energy will be required.

[0101] The origin of the Skelite™ compound and confirmation of themechanism of formation was investigated using techniques which assessthe location of the additive within the HA or TCP structures, in anattempt to observe the presence of the reaction products predicted byequations (3) and (4).

[0102] Significantly, no calcium silicate peaks were identifiable in XRDspectra taken on either colloidal-based or mixed powder compositionswhere Si was the selected additive. This suggests that Si forms adispersed or substituted phase within the phosphate lattice. Previousworkers (Dickens, B. and W. Brown. Acta Cryst B28 1972; pp. 3056-65 andNurse, R., J. Welch and W. Gutt. J Chem Soc 1959; pp. 1077-83 havesuggested that calcium silicate and β-TCP form a miscible solid solutionat high temperatures (>1350° C.) over the composition range of interest.The XRD spectra reported in these earlier experiments did not match thatof α-TCP or the Skelite™ presently described, thus demonstrating theuniqueness of this compound. In this work, when commercially availableCaSiO₃ was physically mixed with cHA or β-TCP powders (Table 1) and thensintered for 8 hr in alumina crucibles in air at 1250° C., the resultsshowed that CaSiO₃ nucleates a crystallographic phase consistent withthe Skelite™ compound (Si-TCP) (FIG. 15). The degree of conversion toSkelite™ increases as the temperature of the reaction is increased. At1250° C. and above, depending on the amount of Si present, the powdermixtures show an increasing tendency to form a melt thus eliminating themicroporous structure.

[0103] Comparison of three major peaks in the XRD spectrum of Skelite™and α-TCP between 2θ_(Cu)=30 and 2θ_(Cu)=31°, assuming a Gaussiantheoretical peak shape with a width of 0.225°, shows that there is ashift of approximately 0.1° to lower 2θ in Si-TCP (FIG. 16) resultingfrom an increase in the lattice parameters. The presence of thissignificant shift was confirmed through the close examination of theposition of the HA peak present in the XRD spectra. The HA peak,2θ_(Cu)=31.8°, was within 0.01° of that predicted by the JCPDS file, andhence the accurate calibration of the instrument was assured. A peakshift in the α-TCP XRD spectra to lower 2θ would occur if Si⁴⁺ (IR=0.26Å for CN=4) substitutes at P⁵⁺ (IR=0.17 Å for CN=4) sites, although theeffect would not be large since the lattice structure is dominated bythe oxygen polyhedra of the TCP. The fact that the substitution reactionoccurs at 1000° C. only for colloidal particles in which the Si ischemically functionalized on the surface suggests that the substitutionkinetics are very slow in the low temperature range.

[0104] Nuclear Magnetic Resonance Studies

[0105] Magic-angle NMR studies were carried out on Si-mHA powders.Comparisons were made with simple physical mixtures of cHA, α- andβ-TCP, CaSiO₃ and SiO₂ in proportions similar to the phases present inthe Si-mHA powders. For Si-mHA, no Si signals could be observed underany conditions of measurement. Careful comparison with signals measuredon CaSiO₃ and amorphous SiO₂ was used to set the lowest level ofsensitivity at which the compounds or local structures could bemeasured. FIG. 17 compares NMR spectra, signal averaged over 120,000pulses, for Si-mHA with that obtained from a simple physical mixture ofcHA and 10% of equal parts of CaSiO₃ and SiO₂. The absence of any NMRsignal in the Si-mHA indicates that Si is highly dispersed throughoutthe crystallographic structure of mHA so that no clearly definablelocation or compound could be identified.

[0106] Infrared Spectroscopy Studies

[0107]FIG. 18 compares infrared spectra for sintered powders of (a) cHA,(b) mHA, and (c) Si-mHA. The peak pair found at the lowest wave numbersnear 600 cm⁻¹ indicate the presence of similar but not identical bonds.The spectra for cHA and mHA powders (no additives) were otherwisegenerally similar. Silicon addition causes a substantial narrowing ofthe P—O stretch peak and a shift in its position from 1048 to 1065 cm⁻¹(FIG. 19).

[0108] In order to assess these changes, IR spectra of CaSiO₃, CaO, SiO₂and commercial β-TCP were examined. The CaSiO₃ spectrum shows a seriesof distinctive peaks at 717, 563 and 434 cm⁻¹ that are not apparentanywhere in the spectra for Si-mHA powders. The CaO spectrum has astrong sequence of bands below 463 cm⁻¹ which are also not observed inthe Si-mHA spectrum. The SiO₂ spectrum shows a very strong,well-resolved peak at 1104 cm⁻¹ characteristic of the Si—O bond. Aninterpretation of the Si-mHA spectra is that the Si—O bond absorptionoccurs at lower wave numbers than in the pure SiO₂. The apparent shiftin the P—O stretch can be explained by the growth of a Si—O peak. It islogical that the Si—O and P—O peaks would occur at similar positionssince silicon and phosphorus are located beside each other in theperiodic table and have similar ionic radii. The fact that the P—O peakappears to shift further indicates the formation of a new siliconcompound, Skelite™.

[0109] A structural model for silicon substitution based on the IRanalysis is a crystal lattice of TCP-like and HA-like material withmolecular dispersion of silicon throughout the lattice. This isconsistent with the NMR and XRD results. The narrowing of the P—O peaksuggests the existence of a less broad distribution of types of P—Obonds within the structure or an increase in crystallinity compared tothe mHA with no introduced additives.

[0110] In Vitro Bone Cell Activity Studies

[0111] The Skelite™ compound on a substrate may be used to assess theresorptive activity of osteoclasts and monitor the change in this levelof resorptive activity either as a result of a disease process or theinclusion, in the culture medium, of an agent such as a drug which wouldinfluence, either directly or indirectly, osteoclastic resorptiveactivity. As provided as a film on a substrate, the compound is alsosuitable for the culture of active osteoblasts in order to observe andassess the secretion of mineralized matrix thereon. As shown in FIG. 20,mineralized collagenous matrix 10 is deposited by cultured osteoblastson the surface of the stabilized thin film 12 as provided on a quartzsubstrate 14. A well integrated boundary layer 16 resembling a cementline is shown which is similar to the same type of cement lines formedby osteoblasts in vivo at the interface between new bone and old bone.This clearly suggests that the biomaterial compound allows forphysiological osteoblast activity further supporting the role of thecompound as an important product that can participate in the naturalbone remodeling process.

[0112] The thin film devices may be used as a means of quantifying theresorptive activity of osteoclasts or the formation of mineralizedmatrix by the activity of osteoblasts. Such activity analysis may occurunder continuous real-time monitoring, time-lapse intervals or end-pointdetermination. The steps in establishing bone cell activity are commonto each of the above monitoring schedules in that bone cells (eitheranimal or human) are cultured, in specific conditions, on one or more ofthe thin film devices. The culture period is from several hours to manydays and preferably from approximately 2 to 10 days (the optimum time iscell species and protocol dependent), during which time the extent ofosteoclast activity may be continuously monitored, periodicallymonitored, or simply not monitored on an on-going basis in favour offinal-end-point determination. FIGS. 11(B) and 22 illustrate osteoclastresorption pits on ceramic pellet and thin film formats of the Si-TCPcompound.

[0113] Similarly, osteoblast activity may be quantified by measuring theamount of mineralized matrix deposition. As is shown in FIG. 21(a), aquartz disc coated with a stabilized film of the present invention andsimultaneously cultured with osteoblasts in the presence of culturemedium containing tetracycline, a natural fluorescent material, displaysfluorescence indicating the presence of mineralized matrix. In contrast,a stabilized film coated on a quartz substrate in the presence of mediumcontaining tetracycline but no osteoblasts (FIG. 21(b)) shows nofluorescence. As the cells take up tetracycline, it is metabolized andincorporated into the newly formed mineralized matrix. The amount ofmineralized matrix is proportional to the measurable fluorescenceemitted. This demonstrates that osteoblasts actively secrete mineralizedmatrix on the stabilized composition.

[0114] The Skelite™ Compound

[0115] The significant correlations with cell-based bioactivity andresistance to dissolution at normal physiological pH 6.4 to 7.3 are thepresence of the additive stabilized compound and the microporousmorphology. The morphology is accounted for by the sintering ofparticles of average size 0.2 to 1.0 μm. The presence of a Si-TCP phasethat is essentially insoluble in biological media at low temperatureusing silicon as the introduced additive is unexpected and is induced bythe distribution of Si substituted throughout the structure. Consideringthat the underlying structure of the particles is the agglomeration ofgranules of size range of approximately 1 to 20 nm, uniform dispersionof the silicon additive and functionalization of the surface of anindividual granule is assured by permeation of the silicon solthroughout the agglomerate. The key aspect of this investigation was thedetermination that silicon does not induce an α-TCP phase resulting fromthe decomposition of HA, but rather it creates a Si-TCP phase, a newbiomaterial compound, by substitution of silicon at phosphorus sites.The fact that silicon induces a Si-TCP compound can now be explainedthrough the crystallography of the calcium-phosphate system and thedefect chemistry associated with silicon substitution into the Ca—Plattice. One skilled in the art would understand that other additiveshaving an ionic radius which is different to that of silicon asdescribed herein, but may still substitute into the Ca—P lattice is alsoembodied for the compound of the present invention. Therefore thecompound is not restricted only to silicon as the additive.

[0116] It is important to note that “effective ionic radius” has beenselected as the term of reference in these studies (Shannon, R. D., ActaCryst. A32., 751, (1976). The ionic radius specifications providedherein reflect the effective ionic radius for coordination numbers of 4,6 or 8. It is apparent to those skilled in the art that “ionic crystalradius” may also be used in the practice of the present invention andthus may be used to define equivalent specifications for the compoundand the formula of the compound as described herein. A summary of theeffective ionic radius and the ionic crystal radius for various elementsis provided in Table 2.

[0117] When substituting Si in the HA lattice, the ionic radius of Si⁴⁺(IR=0.26 Å for CN=4) suggests that Si⁴+can enter at P⁵⁺ (IR=0.17 Å forCN=4) sites within the PO₄ ³⁻ tetrahedra although it could also beincluded at Ca²⁺ (IR=1.0 Å for CN=6) sites. The lattice strain andcompensating defect will be significantly different in the two cases andthe effects of covalency will substantially modify the result. A lowtemperature substitution of Si⁴⁺ into P⁵⁺ sites creates less strain andaccommodates the covalency well. The radius ratio for silicon and oxygenis consistent with that required for the tetrahedral coordination ofsilicon in an oxygen lattice. Such a substitution requires the formationof a single positively charged defect for charge compensation. Anobvious defect is one oxygen vacancy for every two silicon ions,although the energy required to displace oxygen-phosphorous bonds withinan already formed PO₄ ³⁻ tetrahedron may be substantial. Theoretically,substitution of an ion with an appropriate ionic radius and a valence of≧3 at Ca²⁺ sites could also provide charge compensation. Such elementsmay include Ce, La, Sc, Y and Zr. Restrictions on the use of particularelements may be present due to the particular applications for use as abiomaterial.

[0118] In the formation of the Si-TCP compound, compositional analysissuggests that the Ca:P ratio decreases from approximately 1.67 (HA) to1.5 (TCP). This could be induced by (1) the removal of calcium from thelattice, or (2) the introduction of additional phosphorous or an elementthat substitutes for phosphorous. A reduction in the calcium content ofthe lattice could theoretically occur by the formation of calciumsilicate distributed within the structure. However, no evidence ofcalcium silicates as a well defined compound can be found in either theNMR or the IR results. Thus extensive silicon substitution must occurforming a multitude of Si-substituted P—O sites in the lattice.

[0119] In the case of Ti⁴⁺, the ionic radius of (IR=0.42 Å for CN=4)likely precludes its substitution at P⁵⁺ sites and it must thereforeenter the crystal at more general interstitial sites within the lattice.Since titanium has been demonstrated to be less effective in modifyingthe crystal structure to create a stabilized TCP, this suggests that thenucleation of the Si-TCP phase is intimately connected with thesubstitution of silicon at phosphorous sites. In particular, theobserved phase being in fact a Ca—P—Si compound with a crystallographicstructure similar but different from α-TCP rather than pure α-TCP,resolves conflicts with respect to the new compound's decreasedsolubility and the predicted decomposition phase diagram.

[0120] The crystallography of the Ca—P phase diagram has beenextensively studied and compared (Elliott J. Structure and Chemistry ofthe Apatites and Other Calcium Orthophosphates New York: Elsevier (1994)in apatites (Elliott, J. Nature Physical Science 230 1971; p. 72), β-TCP(Dickens, B., L. Schroeder and W. Brown. J Solid State Chem 10 1974; pp.232-48 and Labarther, J., G. Bonel and G. Montel. Ann Chim (Paris) 14thSeries 8 1973; pp. 289-301) and α-TCP (Calvo, C. and R. Gopal. Am Miner60 1975; pp. 120-33). Significant differences have been noted betweenthe structures of α and β-TCP (Elliott J. Structure and Chemistry of theApatites and Other Calcium Orthophosphates New York: Elsevier (1994) andCalvo, C. and R. Gopal. Am Miner 60 1975; pp. 120-33) and equallysignificant similarities have been seen between α-TCP, apatites andcalcium silico-phosphate compounds via the glaserite structure (Mathew,M., W. Schroeder, B. Dickens and W. Brown. Acta Cryst B33 1977; pp.1325-33). A primary component of the phosphate lattice is the presenceof PO₄ ³⁻ tetrahedra, although these structures can vary considerablythroughout a complex lattice. For example, in α-TCP the P—O distancesvary from 1.516 to 1.568 Å and the O—P—O angles vary from 104.1 to115.2° (Calvo, C. and R. Gopal. Am Miner 60 1975; pp. 120-33).Substitution of a Si at such sites implies a range of environments forsuch an additive.

[0121] Following Elliott (Keller, L., P. Rey-Fessler. Characterizationand Performance of Calcium Phosphate Coatings for Implants edited by E.Horowitz and J. Parr. Philadelphia: ASTM, pp. 54-62 (1994) the spacegroup of HA has three kinds of vertical or columnar symmetry. There arecolumns of Ca²⁺ ions spaced by one half of the c-axis parameter alongthree-fold axes which account for two-fifths of the Ca²⁺ ions in thestructure. These ions are given the designation Ca(1). The Ca²⁺ ions arelinked together by PO₄ tetrahedra in which three oxygen atoms come fromone column and the fourth comes from an adjacent column. The result is athree-dimensional network of PO₄ tetrahedra with enmeshed Ca² ⁺ ions,and channels that contain the residual calcium, Ca(2), and ions such asOH⁻ which make up the HA structure.

[0122] The α-TCP structure also comprises columns of Ca²⁺ and PO⁴⁻ ionsparallel to the c-axis (Elliott, J. Nature Physical Science 230 1971; p.72). The columns are actually anion-anion columns . . . CaCaCaCa . . .and cation-anion columns . . . PO₄Ca PO₄ PO₄Ca PO₄ PO₄Ca PO₄ . . . whereis a vacancy (Elliott J. Structure and Chemistry of the Apatites andOther Calcium Orthophosphates New York: Elsevier (1994)). The presenceof this vacancy may facilitate the creation of O²⁻ vacancies inneighboring PO₄ ³⁻ tetrahedra required to accommodate the substitutionof Si⁴⁺ at P5+ sites. Analogous cation-anion columns occur in glaserite,K₃Na(SO₄)₂, except that the vacancy is occupied by a K⁺ ion. Strongsimilarities exist between the glaserite and apatite structures(Dickens, B. and W. Brown. Acta Cryst B28 1972; pp. 3056-65). Theapatite structure can be derived from that of α-TCP by replacingcation-cation columns at the corner of the apatite unit cell by anioncolumns (OH⁻ or F⁻). The remaining cation columns in α-TCP become thecolumnar Ca(1) ions in apatite, whilst the PO₄ ³ ⁻ and Ca²⁺ ions thatform the cation-anion columns in α-TCP have approximately the samepositions as the PO₄ ³⁻ and Ca(2) ions in apatite. Of significance tothis analysis is that the glaserite structure is related tosilico-carnotite Ca₅(PO₄)₂SiO₄ (Labarther, J., G. Bonel and G. Montel.Ann Chim (Paris) 14th Series 8 1973; pp. 289-301) and −Ca₂SiO₄ (Calvo,C. and R. Gopal. Am Miner 60 1975; pp. 120-33). This is consistent withthe report that the system Ca₂SiO₄—Ca₃(PO₄)₂ forms a continuous seriesof solid solutions at higher temperatures based on the glaseritestructure (Nurse, R., J. Welch and W. Gutt. J Chem Soc 1959; pp.1077-83).

[0123] In contrast, there are no such similarities between the structureof HA and β-TCP. The β-TCP structure is a distortion of the parentlattice, Ba₃(VO₄)₂, with layers perpendicular to the c-axis. There is nocolumnar relationship between cations in the structure. Because of thesize of the Ca²⁺ ion, there is a reduction in the number of PO₄tetrahedra in the structure compared to that for the parent lattice anda reduction in the number of formula units within the hexagonal unitcell. Two types of Ca sites exist within the β-TCP unit cell: thoseknown as Ca(5) are fully occupied, while a particular set of cationsites known as Ca(4) are only half occupied (Elliott J. Structure andChemistry of the Apatites and Other Calcium Orthophosphates New York:Elsevier (1994)). Upon doping TCP with Mg²⁺ (IR=0.72 Å for CN=6) the Mgdistributes itself first randomly on the Ca(4) and Ca(5) sites, butsubsequently only substitutes at the Ca(5) sites. Because Mg²⁺ issmaller than Ca²⁺ (IR=1.0 Å for CN=6) and the original distortion of theBa₃(VO₄)₂ structure occurred because Ca²⁺ is smaller than Ba²⁺ (IR=1.35Å for CN=6), the β-TCP structure is stabilized with the addition of Mg²⁺to form the naturally occurring mineral, whitlockite (Calvo, C. and R.Gopal. Am Miner 60 1975; pp. 120-33). Indeed the addition of Mg to β-TCPat high temperatures tends to stabilize the structure well into theα-TCP range. In the case of the addition of an ion such as Ti⁴⁺, theslightly larger ionic radius (IR=0.61 Å for CN=6) would suggest that itwould also be accommodated by substitution at Ca(5) cation sites withresults that are less defined than for Mg²⁺. Since charge compensatingdefects are necessary, the stabilization or creation of Ca²⁺ vacancieson Ca(4) sites would serve this purpose. Therefore substitutional Tishould stabilize the β-phase once TCP has been formed.

[0124] A feature of the presently characterized compound is that theSkelite™ structure is only achieved when intimate contact occurs betweenthe precipitate and the additive. When silicon is introduced intoalready formed and fired powders at relatively low temperatures, theresulting post-sintered phase is predominantly β-TCP. In this case thesilicon plays a role similar to that described for titanium above andsimply acts to reduce the activity of CaO in the decomposition of HAunder the terms of equation (3). In the case of colloidal powdersprecipitated in close association with an additive such as silicon, boththe surface activity will be high and strongly functionalized complexeswill be formed in the solution and at the interfaces of the precipitatedgranules. Through sintering, a range of PO₄ ³⁻ and SiO₄ ⁴⁻ tetrahedrawill be established along with the necessary oxygen vacancies. In thiscase, nucleation of the glaserite-based Si/P phase will take place.While previously this was interpreted as a form of α-TCP it is, in fact,an entirely different compound with its own values for solubility andbioactivity (Si-TCP). Thus the crystal phase composition, surfacemorphology and bulk morphology originates from the chemically active andagglomerated state in which the starting material is precipitated, andthe degree to which this state controls the location at which the Si⁴⁺cation is substituted.

[0125] Again, although silicon has been the most extensively studied andappears to be the preferred substituted element of the invention, it isapparent to one skilled in the art, that any additive that can enter anddistribute throughout the crystal structure of the calcium phosphatelattice and result in the compound of the present invention can besubstituted for silicon. Therefore, the present compound is notrestricted to only silicon as the substituted element but may alsoinclude other suitable elements having a suitable ionic radius ofapproximately 0.1-0.4 Å such as for example boron. It is also understoodthat other additives in addition to the silicon or boron may also bepresent in the compound of the present invention. Such-elements may alsoform part of the Ca—P lattice where such elements and/or the amount ofoxygen may act to balance charge compensation for additives incorporatedinto the compound. Such additives may be selected from the groupconsisting of Ce, La, Sc, Y, and Zr.

[0126] It is also understood by those skilled in the art that the novelcompound of the present invention can be combined with a calciumphosphate material such as calcium hydroxyapatite, α-TCP, β-TCP,octocalcium phosphate, tetracalcium phosphate, dicalcium phosphate,calcium oxide and other like materials. The resultant combination can beas a physical mixture or as a solid solution. In addition, otheradditives such as polymers or microfibers may additionally be added tothe compound of the present invention to increase mechanical strengthand toughness. The particle size of these additives may be selected suchthat the additive may be removed through phagocytosis by the action ofmacrophages. Metals may also be present in combination with the presentcompound to form composite structures. Such structures are also intendedto be embodied in the present invention.

[0127] The Skelite™ Morphology

[0128] The morphology of the synthetic stabilized calcium phosphatecompound (Skelite™) is unique and has not been previously reported ordemonstrated. We have now demonstrated a morphology presenting aninterconnected globular structure of rounded particles having aninterconnected microporosity. In accordance with a preferred aspect ofthis invention, the morphology successfully supports cultures offunctional osteoclasts and osteoblasts.

[0129] The surface morphology of the coating has a characteristic forminvolving a interconnected globular structure (FIG. 6a). The size of theparticles varies from approximately 0.2-1 μm in lateral dimension. Thismorphology may allow for the percolation of liquid media and otherphysiological fluids within the coating. In contrast, the surfacemorphology of hydroxyapatite prepared from other methods, does notresult in a structure as provided by the present invention. In addition,is has been reported that synthetic polycrystalline hydroxyapatite isnot resorbed by osteoclasts (Shimizu, Bone and Minerology, Vol. 6,1989).

[0130] The globular morphology is made up of rounded particlescomparable in size to the aggregated deposits initially made by anosteoblast cell in the process which leads to bone formation. Thepresent composition provides a morphology compatible with the type ofmorphology bone cells encounter in vivo. Particularly, the size andshape of the cell/compound interface facilitates bone cell attachment.Such attachment is a necessary precursor to normal bone cell activity.

[0131] The bulk microporosity of the synthetic stabilized calciumphosphate compound may ensure that the calcium or phosphate ionconcentrations near the surface of the artificial material are withinthe limits expected by the cell as encountered in vivo with natural bonewhich is made up of hydroxyapatite, collagen and other fibrous tissues.During osteoclast mediated extracellular dissolution processes whichlead to resorption, this complex material leads to a particular localconcentration of dissolution products.

[0132] The bioactive synthetic biomaterial ompound of the presentinvention provides a unique chemical composition together with a uniquemorphology and internal microporous structure that has never previouslybeen demonstrated. Compositions have not been previously reported whichdemonstrate consistent bone-cell bioactivity in vivo and in vitro inwhich bioactivity in vitro can be readily, accurately and repetitivelyquantified. The nature of the stabilized biomaterial compound isversatile in that it can be provided in a fine or coarse powder,pellets, three-dimensional shaped pieces, macroporous structures, thinfilms and coatings. In each case, the unique morphology and internalmicroporosity is maintained as well as the stabilized calcium phosphatecomposition.

[0133] In summary, a new calcium phosphate-based biomaterial compoundhas been created and specifically characterized. This new biomaterialexhibits two prominent features:

[0134] (1) A unique composition created by the introduction ofadditives, such as silicon, into the colloidal precipitate to form uponsintering a stabilized calcium phosphate phase comprising the novelcompound.

[0135] (2) A characteristic microporous morphology that arises from theagglomeration of particles within the colloid precipitate and thesintering of the material to produce a network of interconnectedparticles.

[0136] It is now revealed via numerous difficult analytical tests andcomplex data interpretation that this stabilized calcium phosphatecompound is a novel additive stabilized structure referred to asSkelite™ that may exist in combination with HA, α-TCP, β-TCP or othersuitable calcium phosphate phases. This new compound has beencharacterized to have the formula,(Ca_(1-w)A_(w))_(i)[(P_(1-x-y-z)B_(x)C_(y)D_(z)O_(j))]₂, wherein A isselected from those elements having an ionic radius of approximately 0.4to 1.1 Å; B, C and D are selected from those elements having an ionicradius of approximately 0.1 to 0.4 Å; w is greater than or equal to zerobut less than 1; x is greater than or equal to zero but less than 1; yis greater than or equal to zero but less than 1; z is greater than orequal to zero but less than 1; x+y+z is greater than zero but less than1; i is greater than or equal to 2 but less than or equal to 4; and jequals 4-5, where 5 is greater than or equal to zero but less than orequal to 1. The terms w and 5 may be selected to provide chargecompensation of the elements present in the compound.

[0137] An important processing step involves the intimate mixing ofsilicon as a candidate additive with the particles of the colloidalsuspension to ensure the local availability of reactants. This incombination with the similarity of the silicon and phosphorous ionicradii, creates an environment favorable for silicon substitution atphosphorous sites within the Ca—P lattice and the development of thesilicon-stabilized TCP structure.

[0138] The unique composition does not occur in the absence of intimatemixing as the effect of added silicon in these circumstances is only toinfluence the activity of CaO as an HA decomposition product. Similarly,the use of additives comprised of larger ions, such as titanium, cannotbe accommodated in the lattice at phosphorous sites thereby precludingthe important phosphate substitution phenomenon. In both of these cases,the resulting product is predictably β-TCP.

[0139] In view of the ability of Skelite™ to participate in the naturalbone remodeling process, significant opportunities exist for thedevelopment of synthetic bone grafts and bone repair products that areindeed bioactive.

[0140] Synthetic Bone Graft Applications

[0141] A synthetic bone graft that comprises in whole or in part thenovel compound of the present invention has numerous applications in theorthopedic industry. In particular, there are applications in the fieldsof trauma repair, spinal fusion, reconstructive surgery, maxillo-facialsurgery and dental surgery.

[0142] The gold standard in the industry for treating traumatized boneis an autologous bone graft, commonly referred to as an autograft.Autograft transplants involve a surgical procedure in which healthy boneis taken from an alternate part of the patient's skeleton to repairareas of skeletal trauma. Autografts however, require double surgicalprocedures; one for graft removal and a second for re-implantation atthe damaged site. This makes the procedure very expensive and timeconsuming. Additionally, it is not uncommon for patients to subsequentlysuffer chronic pain at the autograft harvest site.

[0143] Another widely used bone graft technique is the use of allograft,a term referring to a tissue graft from another individual or animal. Inthis situation, bone is removed from the donor and implanted in thepatient. Allografts are susceptible to various negative consequences.For example, the use of allograft from an animal other than a humancarries the possibilities of cross species infection and immunologicalrejection. Even human sourced allograft, which is used more often thananimal tissue, exposes the implant recipient to the possibilities ofrejection and disease.

[0144] The use of Skelite™ eliminates the pain and costs associated withthe bone harvest procedure required in autograft transplants.Furthermore, since Skelite™ is generated in a laboratory and iscompletely synthetic, it removes the possibility of transmission ofinfection and disease, as well as eliminates sources of immunologicalrejection by the patient.

[0145] Skelite™ fulfils the need for a versatile bone reconstructionmaterial. Its ability to immediately stimulate local natural bone growthprovides stability and rapid integration, while the body's normalcell-based bone remodeling process slowly resorbs and replaces theimplant with natural bone. This removes the concerns of long termcompatibility and durability associated with current artificial implanttechnologies.

[0146] Products formed from Skelite™ will involve differentconfigurations in order to address the requirements of particularapplications. For example, Skelite™-based products can be manufacturedas a fine or coarse powder, pellets, shaped three dimensional pieces,macroporous structures, thin films and coatings. In addition, theseproducts could potentially carry an integrated bone growth factor tospeed short term recovery.

[0147] The use of Skelite™ in a macroporous configuration allows theopen porous structure to serve as a scaffold for the integration of newbone tissue. The macroporous structure is formed by the coating of thecompound onto a reticulated polymer and subsequently removing thepolymer through pyrolysis. The macroporous structure comprises an opencell construction with interconnected voids having a pore size ofapproximately 50 to 1000 micron. Due to this design, Skelite™ is theideal bone substitute for implantation at defect sites where specialmeasures are required to encourage new bone growth to bridge areas ofmajor tissue loss due to trauma or surgical intervention. The Applicanthas identified two primary approaches for the clinical use of such aproduct: direct implantation and tissue engineering.

[0148] Direct Implantation

[0149] The simplest approach is to directly implant the Skelite™scaffold at the location of skeletal trauma where the bioactiveproperties of the biomaterial compound stimulate the body's natural bonerepair mechanism. Once the initial healing process is complete, theSkelite™ scaffold is progressively replaced with natural bone as part ofthe body's orderly remodeling process.

[0150] Hybrid versions of Skelite™-based products are possible wherebone growth factors are incorporated into the scaffold as apost-manufacturing process or at the time of surgery. The availabilityof the growth factor at the repair site increases the rate of new boneformation thereby improving patient recovery time and lowering overallhealth care costs.

[0151] Tissue Engineering

[0152] The concept that underlies the tissue engineering application isto remove bone cells from the patient's skeleton using an establishedbone marrow aspiration technique, and then carefully introduce thecollected cells (cell seeding) into the open cell structure of theSkelite™ scaffold in a sterile biotechnology facility. The cells andscaffold are then incubated so that the cells have an opportunity tomultiply and begin to fill the scaffold with new mineralized matrix.After several weeks, the biological implant is ready for implantationback into the patient. This biotechnology bone growth process is termed“tissue engineering”, and the procedure serves to enhance the ability ofsurgeons to reconstruct severely compromised areas of the skeleton. Oncesuccessfully integrated at the repair site, the Skelite™ implant issubsequently remodeled into natural bone by the ongoing activity of bonecells.

[0153] A refinement of this approach is to selectively extract andmultiply in cell culture only special precursor cells termed MesenchymalStem Cells (MSCs). In order for these cells to remain healthy duringbiological processing, they need to be attached to a suitable physicalcarrier. In addition, the performance of the cells can benefit from theaddition of organic bone growth factors. Skelite™ is a suitable carriersince it allows for both the integration of bone growth factors and theattachment of specialized MSCs. In addition, following implantation andpatient recovery, the Skelite™ scaffold is subsequently remodeled intonatural bone.

[0154] The use of Skelite™ in direct implantation or tissue engineeringapplications has important advantages over the use of naturally sourcedbone graft material, and consequently Skelite™ products have thepotential to replace the autograft procedure as the orthopedic surgeon'spreferred treatment strategy.

[0155] The key advantages of implantable products formed from theSkelite™ material are:

[0156] Immediately stimulates local natural bone growth at the implantedsite, thus providing early stability and full integration.

[0157] Ensures long term biocompatibility and efficacy.

[0158] Acts as a bioactive scaffold for use in advanced tissueengineering applications.

[0159] Eliminates the cost and chronic pain associated with the doublesurgical procedures required in traditional autograft transplants.

[0160] Eliminates the risks of immunological rejection and infectiontransmission.

[0161] Meets the needs of various orthopedic applications, as theproduct is available in different configurations.

[0162] Allows for the use of growth factors that can further increasethe rate of natural bone healing and subsequent remodeling.

[0163] Provides a means for timed-release drug delivery.

[0164] Disappears naturally through the body's bone remodeling processonce therapeutic function is complete.

[0165] Drug Carrier Application

[0166] The Skelite™ biomaterial may also be used for the incorporationof selected pharmaceuticals into the compound for the furtherenhancement of the bone healing and remodeling processes. In thisrespect, pharmaceuticals that have been incorporated into theSkelite™-based products can be predictably released at the site ofimplantation and hence become available to assist in the boneregeneration process. The Skelite™ biomaterial may also be designed as aslow release vehicle for appropriate pharmaceutical compounds.

[0167] Primary candidates for incorporation into Skelite™-based productsare selected bone growth factors. These proteins have been identified asbeing critically important in growing and maintaining healthy bonetissue. In particular, when applied at the site of traumatized bone,natural bone growth is enhanced with a corresponding improvement inoverall therapeutic response. However, a compatible carrier system isrequired to deliver such therapeutic biologicals to the site and ensurelocal release of appropriate concentrations of the drug. Implant studieshave shown that products formed from the Skelite™ biomaterial aresuitable for use as drug carriers. One skilled in the art wouldunderstand that other pharmaceuticals such as antibiotics for examplewhich may aid in the bone healing process may also be incorporated intothe Skelite™ compound.

[0168] Coating Applications

[0169] Through a liquid application process, the Skelite™ material canbe coated on to orthopedic and dental implants to improve and promotenatural bone fixation and to improve long term implant stability. Such acoating of approximately 0.1 to 10 μm acts at the interface with thepatient's own tissue to promote natural bone growth during the weeksimmediately following surgery, and is then progressively replaced by theongoing activity of bone cells once the initial healing process iscomplete. The result is a strong union between the implant and the hostbone. This is not the case with conventional calcium phosphate implantcoatings where the biologically inert coating is subject to mechanicaldetachment (delamination) from the metal substrate, causing potentiallycatastrophic implant failure.

[0170] The key advantages of an implant coating formed from the Skelite™material are:

[0171] Promotes rapid natural bone growth during the recovery period andis then progressively replaced through the body's orderly remodelingprocess.

[0172] Eliminates the coating as a potential source of long-term failureand reduces the risk to the patient of incurring complicated and costlyrevision surgery.

[0173] Reduces patient recovery time and associated health care costs.

[0174] Permits a strong union directly between the implant and thepatient's natural bone.

[0175] Involves a manufacturing process based on a liquid applicationprocedure which allows full coverage of the device, including complexsurface geometries.

[0176] In Vitro Diagnostic Applications

[0177] As a thin film as provided on a suitable substrate, the Skelite™compound significantly advances the study and understanding of bone cellfunctional properties. The composition and morphology of the stabilizedfilm, as provided in accordance with this invention, permits the cultureof various types of bone cells thereon. The properties of the film maybe adjusted to encourage a significant degree of resorption of theSi-TCP compound of the film material through to a negligible degree ofresorption of the Si-TCP compound in the study of osteoclast activity.Similarly, osteoblast activity may be studied by detecting thedeposition of mineralized matrix. The ability to provide the material ina film format which is sufficiently thin that resorption of the Si-TCPcompound by osteoclasts can be detected provides a simple inexpensiveformat for analysis compared to the prior art techniques. The film asmade in accordance with this invention, supports the biological functionof bone cells. The benefit in providing the film on a transparentsupporting substrate, such as quartz or glass, lends to easy evaluationtechniques of the diagnostic process including automated machinereading.

[0178] Ideally the film thickness is greater than 0.1 micron because ithas been found that at film thicknesses less than 0.1 microns it isdifficult to obtain uniform film coverage, free from discrete voids. Asto the upper thickness limit for the film, it can be of any desiredthickness depending upon its end use. The degree of resorption may bedetected by light transmittance, which preferably requires a film lessthan 10 microns in thickness. The substrate is of quartz which readilywithstands the required sintering temperatures and has the desireddegree of transparency to permit light transmittance tests to determinethe extent of resorption of the film material.

[0179] The developed thin films may be used in kits and analyticalproducts to provide for assessment of bone cell activity. The film maybe embodied in the form of a kit or device comprising quartz substrates,pre-coated with the stabilized calcium phosphate (Si-TCP) compound,which may be used in a cell culture vessel (possibly a 24-welloptionally sterilized multi-well plate i.e. of approximately 15 mmdiameter) as a system suitable for the culture of mixed bone cellpopulations. The device is simple and relies on only routine laboratoryequipment and techniques for use, is suitable for quantitative analysis,and is inexpensive to fabricate but strong enough to withstand normallevels of handling and may be packaged in lots, of (for example) 24samples in a plastic multiwell plate. The thin film surfaces have adefined and reproducible chemistry and are mechanically strong enough towithstand transport when used with an appropriate packing material.

[0180] In each case the culture conditions may be such that osteoclasts,in either mononuclear or multinucleate form could be expected to survivein a functional state and resorb the synthetic stabilized calciumphosphate compound. Similarly, osteoblasts are also capable of activelysecreting mineralized matrix under such culture conditions.

[0181] Once the colloidal suspension (sol-gel) is prepared, it may beapplied as a thin film to the desired substrate in a variety oftechniques. For example, the dip-coating method (C. J. Brinker et al.,Fundamentals of Sol-Gel Dip Coating, Thin Solid Films, Vol. 201, No. 1,97-108, 1991) consists of a series of processes: withdrawal of thesubstrate from a sol-gel or solution at a constant speed, drying thecoated liquid film at a suitable temperature, and firing the film to afinal ceramic.

[0182] In spin-coating the sol-gel is dropped on a plate which isrotating at a speed sufficient to distribute the solution uniformly bycentrifugal action. Subsequent treatments are the same as those of dipcoating.

[0183] It is appreciated that there are a variety of other techniqueswhich may be used to apply a thin film of the sol-gel to the substrate.Other techniques include a spraying of the sol-gel, roller applicationof the sol-gel, spreading of the sol-gel and painting of the sol-gel.

[0184] An alternative to coating discrete discs of a singular size is tocoat an enlarged substrate with a film of the sol-gel. The entire filmon the substrate is then sintered. A device, such as a grid, may then beapplied over the film to divide it into a plurality of discrete testzones.

[0185] In these various techniques of the sol-gel substance application,the thickness and quality (porosity, microstructure, crystalline stateand uniformity) of formed films are affected by many factors. Theseinclude the physical properties, composition and concentration of thestarting sol, the cleanliness of the substrate surface, withdrawal speedof the substrate and the firing temperature. In general the thicknessdepends mainly on the withdrawal rate and sol viscosity for a dipcoating process. Since heterogeneity in the sol-gel is responsible forthe formation of voids, the coating operation should be undertaken in aclean room to avoid particulate contamination of the sol. At theheat-treatment stage, high temperatures are required to develop therequired microstructure and desired conversion of hydroxyapatite intothe biomaterial (Skelite™) compound.

[0186] The purpose of applying the dip coating method to fabricatecalcium phosphate films is twofold: (a) to make films with requiredqualities (uniformity, thickness, porosity, etc.); and (b) to maketranslucent Si-TCP films on transparent substrates for biologicalexperiments.

[0187] Macroporous Structures

[0188] A particular aspect of ceramic preparation for use in biologicalapplications is the fabrication of ceramic pieces with a globularmorphology and internal microporosity which leads to bioactivity, and alarger internal macrostructure of pores of dimensions 50-1000 μm. Thisencourages bone growth and subsequent remodeling in a system moreclosely resembling physiological in vivo bone (FIG. 23). Suchmacroporosity at the low end of the range being particularly suited toin vivo applications desiring rapid ingrowth of bone matrix, whilemacroporosity at the high end of the range allows cells in culture toaccess the interior for uses such as for ex vivo tissue engineeringproduction of bone grafts.

[0189] Using powders with the prefered additive, silicon, and sinteredprior to use, porous ceramics can be made as described herein in theExamples. The procedures described in the accompanying examples resultin the formation of a bulk ceramic having a globular microporousstructure, an underlying internal microporous structure and an internalmacroporous structure allowing cells to migrate and function throughoutthe entire bulk ceramic unit.

[0190] It is to be understood by those skilled in the art that severaldifferent materials and procedures may be used to develop macroporositywithin the ceramic structure. Other materials which are capable ofpyrolysis at temperatures below the normal sintering temperatures arealso useful to form the macroporous structure. The materials used shouldalso not leave any toxic residues. It is also understood that othermethods can also be used to form the macrostructure such as mechanicaldrilling of holes, the use of lasers or use of foaming agents.

[0191] All of the applications in which the present syntheticbiomaterial compound can be used have the advantage that bothosteoclasts and osteoblasts function actively with the compound in anyform thus providing natural cell-mediated remodeling much like thatfound in vivo. The synthetic biomaterial compound of the presentinvention promotes both osteogenesis and resorption so that normaltissue healing can occur while simultaneously allowing the syntheticmaterial to be resorbed in the process of normal bone tissue remodeling.

EXAMPLES

[0192] The examples are described for the purposes of illustration andare not intended to limit the scope of the invention. The examplesexemplify aspects of the invention for providing a Skelite™ compoundwhich is an additive stabilized structure having unique physicalcharacteristics and is fully biocompatible with natural bone tissue.

[0193] Methods of synthetic chemistry and organic chemistry referred tobut not explicitly described in this disclosure and examples arereported in the scientific literature and are well known to thoseskilled in the art.

Example 1 Preparation of Ca—P Colloidal Suspension (Sol-Gel)

[0194] The following procedure is based on preparing sufficient sol-gelhydroxyapatite for manufacturing purposes. Solution A comprises acalcium nitrate tetrahydrate and Solution B comprises an ammoniumdihydrogen orthophosphate (mono basic). Solution A is mixed withSolution B to produce the desired colloidal suspension. Solution A isprepared by adding 40 mls of doubly distilled water to 4.722 grams ofcalcium nitrate, Ca(NO₃)₂. The solution is stirred at moderate speed forsufficient time to dissolve all of the calcium nitrate which is normallyin the range of 3 minutes. To this solution, 3 mls of ammonia hydroxide(NH₄OH) is added and stirred for approximately another 3 minutes. Tothis solution is added 37 mls of double distilled water to provide atotal solution volume of approximately 80 mls. The solution is stirredfor another 7 minutes and covered. The pH of the solution is testedwhere a pH of about 11 is desired.

[0195] Solution B is prepared by adding 60 mls of double distilled waterto a 250 ml beaker containing 1.382 grams of NH₄H₂PO₄. The beaker iscovered and stirred at moderate speed for 3 to 4 minutes until allNH₄H₂PO₄ is dissolved. To this solution is added 71 mls of NH₄OH and thebeaker then covered and stirring continued for approximately another 7minutes. To this is added another 61 mls of double distilled water andthe beaker covered to provide a total solution volume of approximately192 mls. The solution is then stirred for a further 7 minutes andcovered. The pH of the solution is tested where a pH of about 11 isdesired.

[0196] The desired sol-gel is then prepared by combining Solution B withSolution A. All of Solution A is introduced to a 500 ml reagent bottle.Stirring is commenced at a moderate speed and Solution B introduced tothe reagent bottle at a rate of approximately 256 mls per hour until all192 ml of Solution B is delivered into Solution A. An excess of SolutionB may be prepared to compensate for any solution losses which may occurin the transfer process. After completion of this addition andcombination of Solution A with Solution B, the resultant product iscontinued to be stirred at moderate speed for approximately 24 hours.The resultant colloidal suspension (sol-gel) is inspected for anyabnormal precipitation or agglomeration. If any abnormal precipitationor agglomeration has occurred, the solution must be discarded andpreparation commenced again. Approximately 240 mls of the colloidalsuspension, that is the resultant sol-gel, is delivered to a centrifugebottle and centrifuged for 20 minutes at about 500 rpm at roomtemperature. Following centrifugation, 180 mls of supernatant isdiscarded without disturbing the sediments. The sediments are gentlyresuspended by mixing in a smooth rotating manner for about 30 minutes.

[0197] The resulting Ca—P colloidal suspension may be used in a varietyof further preparations.

Example 2 Sintering of Ca—P Products

[0198] The following sintering process may be carried out in standardlaboratory furnaces of various sizes, capable of operating attemperatures from ambient up to at least 1100° C., and designed tomaintain accurate and stable internal temperatures, particularly between800° C. and 1100° C., such as Lindberg models 51744 or 894-Blue M. Thecomponents prepared by any of the procedures described herein arecarefully transferred onto a standard ceramic plate (as is commonpractice in the Lindberg oven). The ceramic plate is used as a carrierduring the sintering process to facilitate easy loading and withdrawalof multiple substrates from the furnace. The furnace temperature is setto the temperature required to achieve the desired ratios of HA:SiTCP.Utilizing a programmable furnace such as the Lindberg model 894-Blue M,the furnace may be programmed to hold the desired temperature, whichwill normally be selected from the range 800° C. to 1100° C., for amaximum of one hour to ensure desired diffusion of the selectedstabilizing additives. The ceramic plate carrying the sinteredsubstrates is removed at any time after the internal furnace temperaturehas cooled to an acceptable and safe touch-temperature of approximately60° C. Individual substrates may then be stored or packaged for finaluse.

[0199] In accordance with this process a fine or coarse powder, pellets,three-dimensional shaped pieces, macroporous structures, thin films andcoatings of the biomaterial compound can be produced on a consistentbasis having the desired composition where variability in the variousprocessing parameters have been minimized to ensure such consistency.

Example 3 Preparation of Thin Films

[0200] To create a thin film on a transparent substrate, quartz(amorphous silica) substrates were cleaned using water and chromic acidand subsequently dip coated in the colloidal suspension of Example 1.The substrate needs to be thoroughly cleaned to ensure satisfactory filmcoverage. In the case of quartz substrates, cleaning is achieved byplacing the discs in a glass beaker and supplying chromic acid cleaningsolution to the glass beaker to cover all discs. The beaker is thencovered. The discs are then sonicated in a water bath for 1 hour. Theacid is washed away using tap water for 20 minutes. The residual tapwater is removed by three changes of doubly distilled water. After thefinal change of double distilled water, every single disc is dried withlint-free towel and inspected for flaws in the quartz surface. Anyresidual particulate on the surface is removed as needed with compressednitrogen or air. The discs are stored in covered trays in an asepticenvironment. This method can be used to clean any type of quartzsubstrate.

[0201] Dip coating was achieved by suction mounting the substrates on acomputer controlled linear slide. The mounted substrates were loweredinto the colloidal suspension and immediately withdrawn at a programmedspeed of 2 mm/s. Following dip coating, the substrates were allowed todry in ambient conditions and were subsequently sintered in aprogrammable furnace for a period of 1 hour at temperatures ranging from800° C. to 1100° C. The sintered thin films had a uniform translucentappearance characteristic of a polycrystalline thin film. The thin filmhad an approximate thickness of 0.5 to 1.0 μm with a particle size onthe order of 0.2 to 1.0 μm.

Example 4 Preparation of Ca—P Powder with No Introduced Additives

[0202] Following the procedures for the formation and aging of thecolloidal suspension of Example 1, the colloid was processed to thestage of reducing the volume by centrifugation. The precipitate wasdried for approximately 5 hours at 100° C. and sintered for one hour inan open alumina crucible in air at a temperature of 1000° C. A finepowder was formed through mechanical grinding of the sintered materialin a motorized mortar and pestle (Retsch Model RM 100 USA).

Example 5 Preparation of Ca—P Powder with Silicon as the IntroducedAdditive

[0203] Following the procedures for the formation and aging of thecolloidal suspension of Example 1, the colloid was processed to thestage of reducing the volume by centrifugation. In order to retain thecolloidal sol characteristics, the silicon additive was introduced as asol-gel metal-organic precursor in an organic carrier. The precursor waseither tetrapropyl orthosilicate (Si(OC₃H,)₄ or TPOS) or tetraethylorthosilicate (Si(OC₂H₅)₄ or TEOS). Addition was accomplished bycreating a sol using a precursor carrier such as 2-methoxyethanol(CH₃OCH₂CH₂OH or 2Me) or 2-4 pentanedione (CH₃COCH₂COCH₃ or ACAC). Theaction of the carrier was to ensure that the additive did notprecipitate upon addition to an aqueous solution having a pH similar tothat of the Ca—P colloidal suspension. This ensured that the additivewas uniformly mixed within the colloid to create a single precipitaterather than two distinct precipitates. Precipitation of the additive wasexamined in a separate experiment with aqueous solutions. For thesilicon compounds, precipitation was minimal for 2Me, ACAC and even ifno carrier was employed. The precipitate with introduced silicon wasdried for approximately 5 hours at 100° C. and sintered for one hour inan open alumina crucible in air at a temperature of 1000° C. A finepowder was formed through mechanical grinding of the sintered materialin a motorized mortar and pestle (Retsch Model RM 100 USA). The presenceof the additive within the sintered ceramics was checked by wet chemicalanalysis.

Example 6 Preparation of Ca—P Powder with Silicon (in the Form of aColloidal Suspension) as the Introduced Additive

[0204] As an alternative to the process defined in Example 5 involvingthe use of a silicon additive in the form of a sol-gel metal-organicprecursor in an organic carrier, a different delivery mechanism forsilicon was utilized where silica was added to the Ca-P colloid by wayof a finely divided (˜10-1100 nm) fumed silica colloid (Cab-o-Sperse™,Cabot Corporation). This approach expands the flexibility of themanufacturing process while providing for intimate mixing between theCa—P colloid and the silica colloid.

[0205] The Ca—P colloid, with the silica colloid added, was subsequentlysprayed into powder using a Yamato-Ohkawara DL-41 spray dryer. Thepowder was then sintered using a heating profile based on a ramp rate of5° C./min., dried at 200° C. for about 180 mins, calcined at 550° C. forabout 60 mins, then sintered at 1000° C. for about 60 mins. The presenceof the additive within the sintered ceramics was confirmed using wetchemical analysis.

Example 7 Preparation of Ca—P Powder with Titanium as the IntroducedAdditive

[0206] Following the procedures for the formation and aging of thecolloidal suspension of Example 1, the colloid was processed to thestage of reducing the volume by centrifugation. In order to retain thecolloidal sol characteristics, the titanium additive was introduced as asol-gel metal-organic precursor in an organic carrier. The precursor wastitanium n-propoxide (Ti(OC₃H₇)₄). Addition was accomplished by creatinga sol using a precursor carrier such as 2-methoxyethanol (CH₃OCH₂CH₂OHor 2Me) or 2-4 pentanedione (CH₃COCH₂COCH₃ or ACAC). ACAC was used inparticular for its strong chelating action. Precipitation of theadditive was examined in a separate experiment with aqueous solutions.For titanium n-propoxide, precipitation of the additive occurred forboth no carrier and 2Me, but not for ACAC. The precipitate withintroduced titanium was dried for approximately 5 hours at 100° C. andsintered for one hour in an open alumina crucible in air at atemperature of 1000° C. A fine powder was formed through mechanicalgrinding of the sintered material in a motorized mortar and pestle(Retsch Model RM 100 USA). The presence of the additive within thesintered ceramics was checked by wet chemical analysis.

Example 8 Preparation of Ceramic Pellets

[0207] Ceramic pellets were formed from previously sintered powder thathad been prepared according to Examples 4, 5, or 6, using a small amountof the concentrated colloid suspension mixed into the sintered powder asa binding agent. The powders were uniaxially pressed into pellets with apressure of 1×10⁸ N/m² [15,000 psi]. The final pellets were sintered forone hour in air at a temperature of 1000° C. to create ceramiccomponents with the desired characteristics. Following thermalprocessing, the pellet density was approximately 1.5 g/cm³, and thepellet exhibited a uniform microporosity throughout the structure.

Example 9 Preparation of Macroporous Structures

[0208] Sintered powder that had been prepared according to Examples 4,5, or 6, was sieved using a motorized sieve shaker (Retsch Model AS200BASIC USA). Powder having a particle size of-325 Mesh was collected andsubsequently suspended in water to form a slurry. The interior andexterior surfaces of a preformed piece of open cell (reticulated)polyurethane foam were completely coated by immersing the foam in theslurry. The slurry-coated component was then allowed to dry and wassubsequently sintered at 1000° C. for 1 hour. During thermal processing,the foam was removed from the structure through pyrolysis. Importantly,the shape of the final ceramic component replicates the original shapeof the foam, including the open-cell structure.

[0209] In the preparation of these components, the pore density of thefoam was selected to produce the required pore size in the ceramic.Typical pore sizes prepared were in the range of 45 to 80 pores perinch. The coating of the foam was managed to ensure complete coverage ofthe foam without clogging of the cells. The duration and temperature ofthe thermal processing were selected to ensure pyrolysis of the foam andto obtain the desired physical properties of the resulting macroporousstructure.

[0210] An alternate method for the formation of a macroporous structureis to introduce styrene balls of a desired size into the powder beingprepared according to Examples 4, 5 or 6. The mixture is combined with abinder, such as a PVA (polyvinyl alcohol) solution, and uniaxiallypressed into pellets. A pressing pressure of 1×10⁸ N/m² [15,000 psi] wasselected so as to not extrude the styrene during compression. Thepellets were subsequently sintered at 1000° C. for one hour during whichthe styrene was removed through pyrolysis.

Example 10 Preparation of Drug Carrier with Associated PharmaceuticalAgent

[0211] Depending on application requirements, either the powder ofExample 5 or the macroporous structure of Example 9 was sterilized usingethylene oxide or similar approved medical device sterilizationtechnique. In a laminar flow hood, a liquid drug volume was made upaccording to dosing requirements. In the case of the agent BCSF™ (BoneCell Stimulating Factor), this required addition of sterile normalsaline (0.9% NaCl) to previously lyophilized stored aliquots of thedrug, at room temperature. Following reconstitution, the drug was eithermixed by gentle agitation with the powder, or slowly dispensed over thesurface of the macroporous structure.

[0212] Recognizing the natural protein avidity of the bioceramicmaterial, a period of 5 minutes was allowed for the drug to percolateand bind to either the powder or the macroporous structure. Followingthis period, the preparation was ready for direct patient administrationas a therapeutic device or for use as a tissue-engineering scaffold.

[0213] In the case of therapeutic administration of the powder-basedpreparation, a predetermined volume of the suspension (powder plusattached pharmaceutical agent) was injected percutaneously at thedesired skeletal site.

[0214] In the case of therapeutic administration of macroporousstructures, surgical intervention was required to implant the device atskeletal sites in order to effect subsequent bone repair.

Example 11 Commercial Reference Materials

[0215] The commercially available HA (cHA), α-TCP, β-TCP, calciumsilicate and silica materials listed in Table 1 (below) were used asreference standards for the analytical techniques performed in theevaluation of the internally prepared mHA and Si-mHA materials describedin this study. TABLE 1 List of Materials Used For Experimental Samplesand Reference Standards Commercial Materials Source Commercial HA cHAAldrich #28,939-6 Lot#04302TQ α-TCP α-TCP Supelco Inc #3-3910 Lot#200792β-TCP β-TCP Fluka #21218 Analysis#357352/1 14996 Calcium silicate CaSiO₃Aldrich #37,266-8 Lot#00714LN Silica SiO₂ PPG Industries Inc. #63231674Lot#9-134 Microporous HA mHA Powder prepared from the thermal processingof the colloid in equation (1) Si-TCP + mHA Si-mHA Powder prepared fromthe thermal processing of the colloid in equation (1) where Si is theintroduced additive

Example 12 Analytical Techniques

[0216] X-ray diffraction (XRD) spectra of thin films were acquired usinga glancing angle (GA-XRD) technique with an angle of incidence θ=2θ,whereas powders were examined using conventional 0-20 geometry. Thesource was a 12 kW Rigaku rotating anode XRD generator fitted with a Crtarget for improved peak resolution. The glancing angle geometrysignificantly reduced the contribution from the substrate. Forconvenience of comparison to other literature, all spectra wereconverted to that expected for a Cu anode using the followingrelationship: sin(θ_(Cu))=(λ_(Cu)/λ_(Cr))sin(θ_(Cr)), whereλ_(Cu)=1.54056 Å and λ_(Cr)=2.28970 Å. The phase composition wasdetermined by comparing acquired spectra with peaks identified in theJoint Committee on Powder Diffraction Standards (JCPDS) database ofstandards [20]. Of particular relevance to this study are the XRDspectra of HA (JCPDS #9-432), α-TCP (JCPDS #9-348) and β-TCP (JCPDS#9-169). Following the collection of XRD data, the background noise wassubtracted and the integrated intensities of peaks distinguishable asHA, α-TCP or β-TCP were calculated. These values were then used todetermine the percentage phase composition (plus or minus 5%).

[0217] Optical microscopy, scanning electron microscopy (SEM, using aJEOL JSM 840) and transmission electron microscopy (TEM, using a PhilipsCM20) were performed to assess the surface and bulk morphology. Chemicalanalysis of the samples was carried out by wet chemical methods andneutron activation analysis. Wide-line nuclear magnetic resonance (NMR)experiments on ²⁹Si were accomplished using a Bruker NMR CXP 200 MHzspectrometer with magic angle spinning using a pulse width of 5 ms and apulse delay of 20 s. Infrared spectroscopy (IR) of powders using a KBrpellet technique utilized a BOMEM MB-120 spectrometer. Approximately 2mg of sample and approximately 200 mg of KBr were ground and pressed ina 6 mm diameter die at 10 tonnes for 1 minute to produce uniform discsfor analysis.

[0218] A particle size analysis of the Ca—P colloid at various stages ofprocessing was made by observation of 633 nm He—Ne laser light scatteredat various angles. Samples were prepared by adding 10 drops of theprecipitated solution to 4 mL ammoniated water (one part 30% NH₄OH mixedwith five parts water) having a pH greater than 10. Results from thesesuspensions were reproducible for equivalent samples and stable overtime. The power spectrum of the scattered light at a known angle wasfitted to a Lorentzian distribution and analyzed by standard methodsusing a solution viscosity of 8.9×10⁴ kg m⁻¹s⁻¹ and refractive index of1.3312 (Clark, N., H. Lunacek and G. Benedek. Am J Phys 38(5) 1970; pp.575-85 and Schumacher, R. Am J Phys 54(2) 1986; pp. 137-41).

[0219] Although preferred embodiments have been described herein indetail, it is understood by those skilled in the art that variations maybe made thereto without departing from the scope of the invention asdefined by the appended claims. TABLE 2 Summary of Effective IonicRadius and Ionic Crystal Radius for Various Elements Data from: Shannon,R. D., Acta Cryst. (1976) A32, 751 Coordination Ionic Crystal EffectiveIonic Ion Number (CN) Radius (CR) Radius (IR) B³⁺ 4 0.25 0.11 6 0.410.27 Ba²⁺ 6 1.49 1.35 8 1.56 1.42 Ca²⁺ 6 1.14 1.00 8 1.26 1.12 Ce³⁺ 61.15 1.01 8 1.28 1.14 La³⁺ 6 1.17 1.03 8 1.30 1.16 Mg²⁺ 4 0.71 0.57 60.86 0.72 8 1.03 0.89 P⁵⁺ 4 0.31 0.17 6 0.52 0.38 Sc³⁺ 6 0.89 0.75 81.01 0.87 Si⁴⁺ 4 0.40 0.26 6 0.54 0.40 Ti⁴⁺ 4 0.56 0.42 6 0.75 0.61 80.88 0.74 Y³⁺ 6 1.04 0.90 8 1.16 1.02 Zr⁴⁺ 4 0.73 0.59 6 0.86 0.72 80.98 0.84

We claim:
 1. A method for the production of a calcium phosphatebiomaterial powder, the method comprising: (a) mixing a silica colloidwith a calcium phosphate colloidal suspension; (b) spray drying (a) intopowder; (c) sintering said powder.
 2. The method of claim 1, whereinsaid silica colloid is finely divided fumed silica colloid.
 3. Themethod of claim 2, wherein said calcium phosphate colloidal suspensionis precipitated from a mixture of calcium nitrate tetrahydrate andammonium dihydrogen orthophosphate.
 4. The method of claim 3, whereinsintering is conducted using a heating profile with a ramp rate of about5° C./minute, drying at about 200° C. for about 180 minutes, calciningat about 550° C. for about 60 minutes, and sintering at about 1000° C.for about 60 minutes.
 5. The method of claim 4, wherein said powder isformed into three dimensional pieces.
 6. The method of claim 5, whereinsaid powder has up to about 10 weight percent of silicon.
 7. A powderedcalcium phosphate biomaterial produced by the method of claim
 1. 8. Amethod for the production of a calcium phosphate biomaterial powder, themethod comprising: (a) mixing a solution of calcium nitrate tetrahydratewith a solution of ammonium dihydrogen orthophosphate to produce acalcium phosphate colloidal suspension; (b) mixing (a) with a finelydivided fumed silica colloid; (c) spray drying (b) into a powder; and(d) sintering using a heating profile with a ramp rate of about 5° C.per minute, drying at about 200° C. for about 180 minutes, calcining atabout 550° C. for about 60 minutes and sintering at about 1000° C. forabout 60 minutes.
 9. A powdered calcium phosphate biomaterial producedby the method of claim
 8. 10. The method of claim 8, wherein saidcalcium phosphate colloidal suspension is centrifuged to reduce thevolume prior to the addition of said finely divided fumed silicacolloid.
 11. The method of claim 2 or 8, wherein said finely dividedsilica colloid has a particle size of about 10-100 nm.